Methods of producing human RPE cells and pharmaceutical preparations of human RPE cells

ABSTRACT

The present invention provides improved methods for producing retinal pigmented epithelial (RPE) cells from human embryonic stem cells, human induced pluripotent stem (iPS), human adult stem cells, human hematopoietic stem cells, human fetal stem cells, human mesenchymal stem cells, human postpartum stem cells, human multipotent stem cells, or human embryonic germ cells. The RPE cells derived from embryonic stem cells are molecularly distinct from adult and fetal-derived RPE cells, and are also distinct from embryonic stem cells. The RPE cells described herein are useful for treating retinal degenerative conditions including retinal detachment and macular degeneration.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a national stage of International Patent ApplicationNo. PCT/US2010/057056 which claims priority to U.S. Provisional PatentApplication No. 61/262,002, filed Nov. 17, 2009, the disclosure of eachof which is herein incorporated by reference.

BACKGROUND OF THE INVENTION

Retinal Pigment Epithelium (RPE)

The retinal pigment epithelium (RPE) is the pigmented cell layer outsidethe neurosensory retina between the underlying choroid (the layer ofblood vessels behind the retina) and overlying retinal visual cells(e.g., photoreceptors—rods and cones). The RPE is critical to thefunction and health of photoreceptors and the retina. The RPE maintainsphotoreceptor function by recycling photopigments, delivering,metabolizing, and storing vitamin A, phagocytosing rod photoreceptorouter segments, transporting iron and small molecules between the retinaand choroid, maintaining Bruch's membrane and absorbing stray light toallow better image resolution. Engelmann and Valtink (2004) “RPE CellCultivation.” Graefe's Archive for Clinical and ExperimentalOphthalmology 242(1): 65-67; See also Irina Klimanskaya, Retinal PigmentEpithelium Derived From Embryonic Stem Cells, in STEM CELL ANTHOLOGY335-346 (Bruce Carlson ed., 2009).

Mature RPE is characterized by its cobblestone cellular morphology ofblack pigmented cells and RPE cell markers including cellularretinaldehyde-binding protein (CRALBP), a 36-kD cytoplasmicretinaldehyde-binding protein that is also found in apical microvilli(Eisenfeld, et al. (1985) Experimental Research 41(3): 299-304); RPE65,a 65 kD cytoplasmic protein involved in retinoid metabolism (Ma, et al.(2001) Invest Opthalmol Vis Sci. 42(7): 1429-35; Redmond (2009) Exp EyeRes. 88(5): 846-847); bestrophin, a membrane localized 68 kD product ofthe Best vitelliform macular dystrophy gene (VMD2) (Marmorstein, et al.(2000) PNAS 97(23): 12758-12763), and pigment epithelium derived factor(PEDF), a 48-kD secreted protein with angiostatic properties(Karakousis, et al. (2001) Molecular Vision 7: 154-163; Jablonski, etal. (2000) The Journal of Neuroscience 20(19): 7149-7157).

Degeneration of the RPE can cause retinal detachment, retinal dysplasia,or retinal atrophy that is associated with a number of vision-alteringailments that result in photoreceptor damage and blindness, such as,choroideremia, diabetic retinopathy, macular degeneration (includingage-related macular degeneration), retinitis pigmentosa, and Stargardt'sDisease (fundus flavimaculatus). WO 2009/051671.

Choroideremia

Choroideremia is an X-linked recessive retinal degenerative disease thatleads to the degeneration of the choriocapillaris, the retinal pigmentepithelium, and the photoreceptor of the eye. Mutations in the CHM gene,which encodes the Rab escort protein-1 (REP-1), cause choroideremia.REP-1 attaches to Rab proteins (involved in intracellular trafficking)and directs the Rab proteins to the organelle membranes. Mutant REP-1proteins cannot escort Rab proteins, leading to a lack of functional Rabproteins. This lack of Rab proteins causes a disruption in intracellulartrafficking and leads to necrosis in the RPE. In childhood, nightblindness is a common first symptom. As the disease progresses, thepatient suffers from a loss of vision, frequently starting as anirregular ring that gradually expands both in toward central vision andout toward the peripheral vision. Genetics Home Reference (U.S. NationalLibrary of Medicine) [Oct. 17, 2010]. Currently, no treatment isavailable and a need exists for a therapy for choroideremia.

Diabetic Retinopathy

Diabetic retinopathy is the most common diabetic eye disease and aleading cause of blindness in the United States. Diabetic retinopathy iscaused by changes in the blood vessels of the retina and occurs in fourstages. First, microaneurysms occur in the retinal blood vessels (MildNonproliferative Retinopathy). As the disease progresses, blood vesselsbecome blocked leading to Moderate Nonproliferative Retinopathy. As moreblood vessels are blocked this deprives several areas of the retina oftheir blood supply (Severe Nonproliferative Retinopathy.) Finally,signals sent by the retina for nourishment trigger the growth of newblood vessels (proliferative retinopathy) but these new blood vesselsare abnormal and fragile. The new abnormal blood vessels grow along theretina and along the surface of the vitreous humour inside of the eye.As the structural integrity of the blood vessels deteriorate (in partdue to changes in osmolarity due to insulin/sugar imbalance fundamentalto diabetes), they leak blood, causing severe vision loss and evenblindness. “Diabetic Retinopathy” (MayoClinic.org) [Feb. 11, 2010].Generally, diabetic retinopathy may only be controlled or slowed withsurgery but not treated, and the patient usually continues to sufferfrom vision problems. Therefore, there exists a need for improveddiabetic retinopathy therapies.

Macular Degeneration

Age-related macular degeneration (AMD) is the most common reason forlegal blindness in the United States and Europe. Atrophy of thesubmacular RPE and the development of choroidal neovascularizations(CNV) results secondarily in loss of central visual acuity. Early signsof AMD are deposits (druses) between retinal pigment epithelium andBruch's membrane. Central geographic atrophy (“dry AMD”) results fromatrophy to the retinal pigment epithelial layer below the retina, whichcauses vision loss through loss of photoreceptors (rods and cones) inthe central part of the eye. Neovascular or exudative AMD (“wet AMD”)causes vision loss due to abnormal blood vessel growth (choroidalneovascularization) in the choriocapillaris, through Bruch's membrane,ultimately leading to blood and protein leakage below the macula.Bleeding, leaking, and scarring from these blood vessels eventuallycause irreversible damage to the photoreceptors and rapid vision loss ifleft untreated. Current treatments for macular degeneration includeanti-angiogenic therapy with ranibizumab (LUCENTIS®) or bevacizumab(AVASTIN®), photocoagulation (laser surgery), photodynamic therapy withverteporfin (VISUDYNE®), and submacular hemorrhage displacement surgery.“Macular Degeneration.” (MayoClinic.org) [October 2010]. However, thegoal of these therapies is to stem further vision loss and,unfortunately, existing damage cannot be reversed. Therefore, a greatneed exists for the treatment of macular degeneration.

Retinitis Pigmentosa (RP)

Retinitis pigmentosa (RP) is a group of inherited diseases that damagethe photoreceptors (e.g., rods and cones) in the retina affectingapproximately 1.5 million people worldwide. For example, autosomalrecessive RP is caused by mutations in cis retinaldehyde binding proteinor RPE65. The progression of RP is slow and varies from patient topatient. Patients with RP all suffer some vision loss, with nightblindness as a typical early symptom followed by tunnel vision, and somemay lose all sight. “Retinitis Pigmentosa.” American OptometricAssociation (October 2010). Although treatment with vitamin A and luteinhas shown some promise in slowing the progress of RP, no effectivetreatment is available.

Retinal Detachment

Retinal detachment, including rhegmatogenous retinal detachment,exudative, serous, or secondary retinal detachment, and tractionalretinal detachment, is a disorder of the eye in which the retina peelsaway from its underlying layer of support tissue. Initial detachment maybe localized, but without rapid treatment the entire retina may detach,leading to vision loss and blindness. See Ghazi and Green (2002) Eye 16:411-421. A minority of retinal detachments arise from trauma includingblunt blows to the orbit, penetrating trauma, and concussions. Thecurrent treatment is emergency eye surgery but only has an approximately85% success rate, and even if successful, the patient may suffer a lossof visual acuity and visual artifacts. See Facts About RetinalDetachment [NEI Health Information] (October 2010). Therefore, a needexists for a treatment for retinal detachment.

Stargardt's Disease (Fundus Flavimaculatus)

Stargardt's Disease (fundus flavimaculatus) is a type of maculardegeneration, including both an autosomal recessive and a dominant form,that causes a progressive loss of central vision of both eyes, but doesnot affect peripheral vision. Patients with Stargardt's experience agradual deterioration of the retina's cone receptor cells. Cones areconcentrated in the macula, and are responsible for central vision andcolor. Over time, these diseased cells cause a blackened hole to form inthe central vision, and the ability to perceive colors is eventuallyaffected. See Gass and Hummer (1999) Retina 19(4): 297-301 and Aaberg(1986) Tr. Am. Ophth. Soc. LXXXIV: 453-487. Currently, there are notreatments available for Stargardt's Disease.

RPE Cells in Medicine

Given the importance of the RPE in maintaining visual and retinalhealth, the RPE and methodologies for producing RPE cells in vitro areof considerable interest. See Lund, et al. (2001) Progress in Retinaland Eye Research 20(4): 415-449. For example, a study reported inGouras, et al. (2002) Investigative Ophthalmology & Visual Science43(10): 3307-311 describes the transplantation of RPE cells from normalmice into transgenic RPE65^(−/−) mice (a mouse model of retinaldegeneration). Gouras discloses that the transplantation of healthy RPEcells slowed the retinal degeneration in the RPE65^(−/−) mice but after3.7 weeks, its salubrious effect began to diminish. Treumer, et al.(2007) Br J Opthalmol 91: 349-353 describes the successfullytransplantation of autologous RPE-choroid sheet after removal of asubfoveal choroidal neovascularization (CNV) in patients with agerelated macular degeneration (AMD), but this procedure only resulted ina moderate increase in mean visual acuity.

Moreover, RPE cells have been suggested as a possible therapy fortreating Parkinson's disease, a chronic degenerative disease of thebrain. The disease is caused by degeneration of specialized neuronalcells in the region of the basal ganglia. The death of dopaminergicneurons results in reduced synthesis of dopamine, an importantneurotransmitter, in patients with Parkinson's disease. The standardtherapy is medical therapy with L-dopa. L-dopa is metabolized in thebasal ganglia to dopamine and there takes over the function of themissing endogenous neurotransmitter. See McKay, et al. (2006) ExpNeurol. 20(1): 234-243 and NINDS Parkinson's Disease Information Page(Sep. 23, 2009). However, L-dopa therapy loses its activity after someyears, and thus, a new therapy for Parkinson's disease is needed. Forexample, Ming and Le (2007) Chinese Medical Journal 120(5): 416-420suggests the transplantation of RPE cells from eye donors into thestriatum of Parkinson's patients to supply beneficial neurotrophic andanti-inflammatory cytokines to treat Parkinson's′ disease.

However, RPE cells sourced from human donors has several intractableproblems. First, is the shortage of eye donors, and the current need isbeyond what could be met by donated eye tissue. For example, RPE cellssourced from human donors are an inherently limited pool of availabletissue that prevent it from scaling up for widespread use. Second, theRPE cells from human donors may be contaminated with pathogens and mayhave genetic defects. Third, donated RPE cells are derived fromcadavers. The cadaver-sourced RPE cells have an additional problem ofage where the RPE cells are may be close to senesce (e.g., shortertelomeres) and thus have a limited useful lifespan followingtransplantation. Reliance on RPE cells derived from fetal tissue doesnot solve this problem because these cells have shown a very lowproliferative potential. Further, fetal cells vary widely from batch tobatch and must be characterized for safety before transplantation. See,e.g., Irina Klimanskaya, Retinal Pigment Epithelium Derived FromEmbryonic Stem Cells, in STEM CELL ANTHOLOGY 335-346 (Bruce Carlson ed.,2009). Any human sourced tissue may also have problems with tissuecompatibility leading to immunological response (graft-rejection). Also,cadaver-sourced RPE cells may not be of sufficient quality as to beuseful in transplantation (e.g., the cells may not be stable orfunctional). Fourth, sourcing RPE cells from human donors may incurdonor consent problems and must pass regulatory obstacles, complicatingthe harvesting and use of RPE cells for therapy. Fifth, a fundamentallimitation is that the RPE cells transplanted in an autologoustransplantation carry the same genetic information that may have lead tothe development of AMD. See, e.g., Binder, et al. (2007) Progress inRetinal and Eye Research 26(5): 516-554. Sixth, the RPE cells used inautologous transplantation are already cells that are close to senesce,as AMD may develop in older patients. Thus, a shorter useful lifespan ofthe RPE cells limits their utility in therapeutic applications (e.g.,the RPE cells may not transplant well and are less likely to last longenough for more complete recovery of vision). Seventh, to be successfulin long-term therapies, the transplanted RPE cells must integrate intothe RPE layer and communicate with the choroid and photoreceptors.Eighth, in AMD patients and elderly patients also suffer fromdegeneration of the Bruch's membrane, complicating RPE celltransplantation. See Gullapalli, et al. (2005) Exp Eye Res. 80(2):235-48. Thus there exists a great need for a source of RPE cells fortherapeutic uses.

Embryonic Stern Cells Derived RPE Cells (hESC-RPE Cells)

Human embryonic stem cells (hES) are considered a promising source ofreplacement RPE cells for clinical use. See Idelson, et al. (2009) CellStem Cell 5: 396-408. However, numerous problems continue to plaguetheir use as therapeutics, including the risk of teratoma-formation andthe need for powerful immunosuppressive drugs to overcome the problemswith immune rejection. For example, Wang, et al. (2010) Transplantationdescribes a study where mouse embryonic stem cells were differentiatedinto RPE cells and then transplanted into a mouse model of retinitispigmentosa (Rpe65^(rd12)/Rpe^(rd12) C57BL6 mice). Although theRpe65^(rd12)/Rpe^(rd12) mice receiving the RPE cell transplants did showsignificant visual recovery during a 7-month period, this wascomplicated by retinal detachments and tumors.

Further, the transition from basic research to clinical application isprecluded by the need to adhere to guidelines set forth by the U.S. Foodand Drug Administration, collectively referred to as current GoodManufacturing Practices (GMP) and current Good Tissue Practices (GTP).In the context of clinical manufacturing of a cell therapy product, suchas hES cell-derived RPE, GTP governs donor consent, traceability, andinfectious disease screening, whereas the GMP is relevant to thefacility, processes, testing, and practices to produce a consistentlysafe and effective product for human use. Lu, et al. Stem Cells 27:2126-2135 (2009). Thus, there exists a need for a systematic, directedmanner for the production of large numbers of RPE cells suitable for usein transplantation therapies.

SUMMARY OF THE INVENTION

The present invention provides methods for differentiating RPE cellsfrom pluripotent stem cells. The present invention also providesfunctional retinal pigmented epithelial cells (RPE) that are terminallydifferentiated from pluripotent stem cells. These methods may be used toproduce large numbers of functional differentiated RPE cells for use intherapeutic methods (and uses), screening assays, and to study the basicbiology of the RPE. The present invention also provides preparationsincluding pharmaceutical preparations of RPE cells derived frompluripotent stem cells.

In one embodiment, the invention provides a method of producing asubstantially purified culture of retinal pigment epithelial (RPE) cellscomprising

-   -   (a) providing pluripotent stem cells;    -   (b) culturing the pluripotent stem cells to form embryoid bodies        in nutrient rich, low protein medium;    -   (c) culturing the embryoid bodies to form an adherent culture in        nutrient rich, low protein medium;    -   (d) culturing the cells of (c) in medium capable of supporting        growth of high-density somatic cell culture, whereby RPE cells        appear in the culture of cells;    -   (e) dissociating the culture of (d);    -   (f) selecting the RPE cells from the culture and transferring        the RPE cells to a separate culture containing medium        supplemented with a growth factor to produce an enriched culture        of RPE cells; and    -   (g) propagating the enriched culture of RPE cells to produce a        substantially purified culture of RPE cells.

In another embodiment, the invention provides a method of producing asubstantially pure culture of mature retinal pigment epithelial (RPE)cells comprising

-   -   (a) providing pluripotent stem cells;    -   (b) culturing the pluripotent stem cells to form embryoid bodies        in nutrient rich, low protein medium;    -   (c) culturing the embryoid bodies to form an adherent culture in        nutrient rich, low protein medium;    -   (d) culturing the cells of (c) in medium capable of supporting        growth of high-density somatic cell culture, whereby RPE cells        appear in the culture of cells;    -   (e) dissociating the culture of (d);    -   (f) selecting the RPE cells from the culture and transferring        the RPE cells to a separate culture containing medium        supplemented with a growth factor to produce an enriched culture        of RPE cells;    -   (g) propagating the enriched culture of RPE cells; and    -   (h) culturing the enriched culture of RPE cells to produce        mature RPE cells.

In one embodiment, the pluripotent stem cells are embryonic stern cells,induced pluripotent stem (iPS) cells, adult stem cells, hematopoieticcells, fetal stem cells, mesenchymal stem cells, postpartum stem cells,multipotent stem cells, or embryonic germ cells. In another embodiment,the pluripotent stem cells may be mammalian pluripotent stem cells. Instill another embodiment, the pluripotent stern cells may be humanpluripotent stem cells including but not limited to human embryonic stem(hES) cells, human induced pluripotent stem (iPS) cells, human adultstem cells, human hematopoietic stem cells, human fetal stem cells,human mesenchymal stem cells, human postpartum stem cells, humanmultipotent stem cells, or human embryonic germ cells. In anotherembodiment, the pluripotent stem cells may be a hES cell line listed inthe European Human Embryonic Stem Cell Registry—hESCreg.

In one embodiment, the present invention provides preparations of RPEcells, including substantially purified preparations of RPE cells.Exemplary RPE cells may be differentiated from pluripotent stem cells,such as embryonic stem cells, iPS cells, blastomeres, inner mass cells,or oocytes which may be parthenogenetically activated. These pluripotentstem cells may be recombinant or genetically engineered (e.g.,engineered to express a desired therapeutic protein or to eliminate theexpression of a gene involved in a genetic deficiency such as maculardegeneration.) The RPE cells may be formulated and used to treat retinaldegenerative diseases. Additionally, pluripotent stem cell-derived RPEcells can be used in screening assays to identify agents that modulateRPE cell survival (in vitro and/or in vivo), to study RPE cellmaturation, or to identify agents that modulate RPE cell maturation.Agents identified using such screening assays may be used in vitro or invivo and may provide additional therapeutics that can be used alone orin combination with RPE cells to treat retinal degenerative diseases.

In one embodiment, the pluripotent stem cells of (a) may be geneticallyengineered.

In one embodiment, the medium of (a), (b), (c), (d), (f), (g), or (h)contains serum free B-27 supplement. In another embodiment, the mediumof (a), (b), (c), (d), (I), (g), or (h) does not contain serum free B-27supplement.

In one embodiment, the cells of (b) are cultured for at least about 7-14days. In another embodiment, the cells of (c) are cultured for at leastabout 7-10 days. In a further embodiment, cells of (e) are cultured forat least about 14-21 days.

In one embodiment, the medium of (a), (b), (c), (d), (f), (g), or (h) isMDBK-GM, OptiPro SFM, VP-SFM, EGM-2, or MDBK-MM. In another embodiment,the growth factor of (f) is EGF, bFGF, VEGF, or recombinant insulin-likegrowth factor. In a further embodiment, the medium (g) comprisesheparin, hydrocortisone, or ascorbic acid. In yet another embodiment,the culture medium used for propagating the enriched culture of RPEcells does not support the growth or maintenance of undifferentiatedpluripotent stem cells.

In one embodiment, step (e) comprises contacting the culture with anenzyme selected from the group consisting of trypsin, collagenase,dispase, papain, mixture of collagenase and dispase, and a mixture ofcollagenase and trypsin. In another embodiment, step (e) comprisesmechanical disruption.

In one embodiment, the pluripotent stem cells have reduced HLA antigencomplexity.

In one embodiment, the method further comprising culturing said RPEcells under conditions that increase alpha integrin subunit expression,wherein said alpha integrin subunits are 1-6 or 9. In anotherembodiment, the conditions comprising exposure to manganese, exposure toan antibody to CD29, or passaging said RPE cells for at least about 4passages. In a further embodiment, the anti-CD29 antibody is monoclonalantibody HUTS-21 or monoclonal antibody (mAb) TS2/16.

In one embodiment, the invention provides a pharmaceutical preparationof RPE cells suitable for treatment of retinal degradation, wherein saidRPE cells have at least one of the following properties:

-   -   (a) maintain their phenotype after transplantation for at least        about one month,    -   (b) maintain their phenotype in culture for at least about one        month,    -   (c) integrate into the host after transplantation,    -   (d) do not substantially proliferate after transplantation,    -   (e) are phagocytositic,    -   (f) deliver, metabolize, or store vitamin A,    -   (g) transport iron between the retina and choroid after        transplantation,    -   (h) attach to the Bruch's membrane after transplantation,    -   (i) absorb stray light after transplantation,    -   (j) have elevated expression of alpha integrin subunits, or    -   (k) have longer telomeres than RPE cells derived from human        donors.        In another embodiment, the RPE cells have at least 1, 2, 3, 4,        5, or 6 of the recited properties. In yet another embodiment,        the RPE cells are phagocytositic and have longer telomeres than        RPE cells derived from human donors.

In one embodiment, the invention provides a pharmaceutical preparationfor use in treating retinal degeneration comprising an effective amountof RPE cells. In another embodiment, the retinal degeneration is due toStargardt's disease, age-related macular degeneration (AMD),choroideremia, retinitis pigmentosa, retinal detachment, retinaldysplasia, or retinal atrophy.

In one embodiment, the pharmaceutical preparation of RPE cells isformulated for transplantation in the form of a suspension, gel, orcolloid. In another embodiment, the preparation is formulated fortransplantation with a matrix, substrate, scaffold, or graft. In afurther embodiment, the preparation is formulated for administration tothe subretinal space of the eye. In a further embodiment, thepreparation comprises at least about 10³-10⁹ RPE cells.

In one embodiment, the RPE cell preparation comprises mature RPE cells.In another embodiment, the RPE cell preparation consist essentially ofmature RPE cells. In a further embodiment, the preparation comprises atleast about 75% RPE cells.

In one embodiment, the preparation is substantially free of viral,bacterial, and/or fungal contamination. In another embodiment, thepreparation is formulated in a pharmaceutically acceptable carrier. In afurther embodiment, the preparation is formulated for administration tothe eye. In a still further, the preparation is formulated foradministration to the sub-retinal space. In another embodiment, the RPEcells are functional RPE cells capable of integrating into the retinaupon transplantation. In another embodiment, the preparation issubstantially free of mouse embryo fibroblasts (MEF) and human embryonicstem cells (hES). In a further embodiment, the preparation is GoodManufacturing Practices (GMP) compliant.

In one embodiment, the invention provides a cryopreserved preparationcomprising at least about 10⁴ human RPE cells, wherein the preparationis a substantially purified preparation of human RPE cells derived fromhuman pluripotent stem cells, and wherein the RPE cells express RPE-65,Bestrophin, PEDF, CRALBP, Otx2, and Mit-F. In another embodiment, atleast about 85% of the RPE cells retain viability following thawing.

In one embodiment, the invention provides a substantially purifiedpreparation of human RPE cells differentiated from human pluripotentstem cells, wherein the RPE cells express, at the mRNA and proteinlevel, RPE-65, Bestrophin, PEDF, CRALBP, Otx2, and Mit-F, and whereinthe cells substantially lack expression of Oct-4, NANOG, and Rex-1. Inanother embodiment, the RPE cells comprise differentiated RPE cells andmature differentiated RPE cells, and wherein at least the maturedifferentiated RPE cells further express, at the mRNA and protein level,PAX2, pax-6, and tyrosinase. In another embodiment, the RPE cells aredifferentiated from human ES cells or human IPS cells.

In one embodiment, the invention provides for the use of apharmaceutical preparation of RPE cells in the manufacture of amedicament for the treatment of retinal degeneration.

In one embodiment, the invention provides a method of cryopreserving RPEcells comprising

-   -   (a) culturing RPE cells,    -   (b) harvesting said RPE cells,    -   (c) centrifuging said RPE cells, and    -   (d) resuspending said RPE cells in 10% DMSO/90% FBS solution.

In one embodiment, the RPE cells are washed with Ca²⁺/Mg⁺ DPBS. Inanother embodiment, the RPE cells were cultured until bestrophin isorganized at the cell membrane. In another embodiment, the RPE cells arecultured until they reach a medium pigmentation level. In anotherembodiment, step (a) comprising culturing at least two culture vesselsof RPE cells. In another embodiment, the RPE cells are harvested andcombined into a single lot. In another embodiment, the RPE cells areharvested and stored in FBS during the combination of RPE cells.

In one embodiment, the invention provides a method of treating retinaldegeneration comprising a pharmaceutical preparation comprisingadministering an effective amount of RPE cells described herein. Inanother embodiment, the retinal degeneration is due to choroideremia,diabetic retinopathy, age-related macular degeneration, retinaldetachment, retinitis pigmentosa, or Stargardt's Disease.

In one embodiment, the preparation is transplanted in a suspension,matrix, gel, colloid, scaffold, or substrate. In another embodiment, thepreparation is administered by injection into the subretinal space ofthe eye.

In a further embodiment, the effective amount is at least about20,000-200,000 RPE cells. In another embodiment, the effective amount isat least about 20,000, 50,000, 75,000, 100,000, 125,000, 150,000,175,000, 180,000, 185,000, 190,000, or 200,000 RPE cells.

In one embodiment, the method further comprising monitoring the efficacyof the method by measuring electroretinogram responses, optomotor acuitythreshold, or luminance threshold in the subject.

In one embodiment, the preparation is substantially free of viral,bacterial, or fungal contamination. In another embodiment, the RPE cellsare functional RPE cells capable of integrating into the retina upontransplantation. In a further embodiment, the RPE cells improve visualacuity following transplantation.

The present invention provides methods for the treatment of eyedisorders. In particular, these methods involve the use of RPE cells totreat or ameliorate the symptoms of eye disorders, particularly eyedisorders caused or exacerbated, in whole or in part, by damage to orbreakdown of the endogenous RPE layer (e.g., retinal degeneration).

In one embodiment, the RPE cells described herein are substantially freeof genetic mutations that may lead to retinal degeneration.

In one embodiment, the RPE cells may be transplanted with abiocompatible polymer such as polylactic acid, poly(lactic-co-glycolicacid), 50:50 PDLGA, 85:15 PDLGA, and INION GTR® biodegradable membrane(mixture of biocompatible polymers).

In another embodiment, the RPE cells adhere to Bruch's membrane aftertransplantation, establish polarity, and integrate into the receipt'stissue.

In one embodiment, the RPE cells may improve visual acuity aftertransplantation. In another embodiment, the RPE cells may substantiallyimprove visual acuity after transplantation.

In one embodiment, the RPE cells may be in compliance with at least oneof the GTP and/or GMP Regulations as presented in Table 3 or 4. Inanother embodiment, the RPE cells may be produced in accordance withGood Manufacturing Practice (GMP). In a further embodiment, the RPEcells may be produced in accordance with Good Tissue Practice (GTP). Ina further embodiment, the RPE cells may meet at least one of thecriteria recited in Table 4. In a still further embodiment, the RPEcells may meet at least 1, 2, 3, 4, or 5 of the criteria recited inTable 4.

In one embodiment, the RPE cells lack substantial expression ofembryonic stem cell markers including but not limited to Oct-4, NANOG,Rex-1, alkaline phosphatase, Sox2, TDGF-1, DPPA-2, and DPPA-4. Inanother embodiment, the RPE cells express RPE cell markers including butnot limited to RPE65, CRALBP, PEDF, Bestrophin, MitF, Otx2, PAX2, Pax-6,and tyrosinase. In a further embodiment, the RPE cells express at leastone of the genes listed in Table 5, and wherein expression of the atleast one gene is increased in the RPE cells relative to expression inhuman ES cells. In a still further embodiment, the RPE cells express atleast one of the genes listed in Table 6, and wherein expression of theat least one gene is decreased in the RPE cells relative to expressionin human ES cells. In one embodiment, the RPE cells show increased alphaintegrin subunit expression. In another embodiment, the alpha integrinsubunit is alpha 1, 2, 3, 4, 5, 6, or 9. In yet another embodiment, theexpression is mRNA expression, protein expression, or both mRNA andprotein expression.

The present invention provides for a method of providing a RPEpreparation to a clinical site comprising (a) thawing vials ofcryopreserved RPE cells, (b) resuspending the RPE cells in media, (c)centrifuging the RPE cells, (d) resuspending the RPE cells in media, (e)aliqouting the RPE cells into vials, and (f) transferring to theclinical site. In one embodiment, the resuspension and centrifugationsteps may be repeated at least 1, 2, 3, 4, or 5 times. In anotherembodiment, the RPE product is transported to the clinical site withinat least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours of completion ofstep (e). In a further embodiment, the vials may be labeled.

The present invention also provides a method for a providing RPE cellpreparation for sale comprising (a) producing RPE cells and (b)preparing said RPE cell preparations for transfer to a customer. In oneembodiment, the method may comprise cryopreserving the RPE cells. Inanother embodiment, the method comprises offering said RPE cellpreparations for sale. In a further embodiment, the method comprisesadvertising the RPE cell preparations.

The invention contemplates any combination of the aspects andembodiments described above or below. For example, preparations of RPEcells comprising any combination of differentiated RPE cells and matureRPE cells can be used in the treatment of any of the conditionsdescribed herein. Similarly, methods described herein for producing RPEcells using human embryonic stem cells as a starting material may besimilarly performed using any human pluripotent stem as a startingmaterial.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-7 depict exemplary protocols for the production of RPE cells.

FIG. 1 Production of RPE Cells: Step 1—Preparation of MEF Feeder Cells.The MEF feeder cells may be cultured in the presence of about 10-20nm/mL human leukemia inhibitory factor (LW) and about 8-16 ng/mL humanbFGF. See, e.g., Irina Klimanskaya, Retinal Pigment. Epithelium DerivedFrom Embryonic Stem Cells, in S TEM C ELL A NTHOLOGY 335-346 (BruceCarlson ed., 2009).

FIG. 2 Production of RPE Cells: Step 2—Seeding and Expansion of hESCells.

FIG. 3 Production of RPE Cells: Step 3—Embryoid Body Formation.

FIG. 4 Production of RPE Cells: Step 4—RPE Derivation. Clusters of RPEcells may appear within 6-8 weeks, where RPE cells may appear on thesurface of the embryoid bodies and then slowly spread to the entireembryoid body over time.

FIG. 5 Production of RPE Cells: Step 5—RPE Expansion andDifferentiation. In one embodiment, the RPE cell cultures may be washedat least 1, 2, 3, 4, or 5 times to remove loose or isolated cells. Theinventors found that this surprisingly improved the yield of RPE cells.The RPE cells may be characterized by the expression of RPE-specificcell markers such as CRALBP, bestrophin, RPE65, and PEDF. The RPE cellsmay also be characterized by functional tests including a RPE-specificphagocytosis assay and vitamin A metabolism assay. See, e.g., IrinaKlimanskaya, Retinal Pigment Epithelium Derived From Embryonic SteinCells, in STEM CELL ANTHOLOGY 335-346 (Bruce Carlson ed., 2009).

FIG. 6 Production of RPE Cells: Step 6—Harvest, Culturing, andCryopreservation. In one embodiment, several flasks of RPE cells may beseeded and propagated to yield a large amount of RPE cells. Asindividual flasks of RPE cells are harvested (e.g., T-75 flasks), theRPE cells may be stored in FBS at about 4° C. during the harvestingsteps. Additionally, the RPE cells may be considered ready forcryopreservation when the dystrophin is organized at the cell membraneand the PAX6 expression is low. The inventors found that thissurprisingly improved the viability of the cryopreserved RPE cells.

FIG. 7 Production of RPE Cells: Step 7—Thawing of Cryopreserved RPEcells and Pharmaceutical Preparation.

FIG. 8 depicts the Log up- or downregulation of ES and RPE markers,respectively, in RPE cells. The mean±SD relative gene expression ofseven representative lots of RPE are shown. Data have been normalized toβ-actin control levels for each sample and are expressed relative to thelevels of expression observed in MA09 hES cells. The four upregulatedRPE markers (e.g., RPE-65, PAX6, Bestrophin, and MIT) are shown on theleft; the three downregulated hES markers (e.g., OCT4, NANOG and SOX2)are shown on the right.

FIG. 9 depicts electrical activity of the outer (a-wave) and inner(b-wave) retina in response to light flashes test by ERG responses atboth P60 and P90. ERG responses in RPE grafted animals achievedsignificantly better responses over sham controls (p<0.05, t-test).

FIG. 10 depicts date from an optomoter data system shows that shows thatthe RPE treated eyes performed significantly better than thesham-treated and untreated eyes (p<0.05, t-test), giving approximately50% and 100% improvement in visual acuity over the sham and untreatedcontrols, respectively.

FIG. 11 Luminance threshold at P100—luminance threshold responsesrecorded across the superior colliculus, each curve (average SEM) showsthe percent of retinal area (y-axis) where the visual threshold is lessthan the corresponding value on the x-axis (log units, relative tobackground illumination 0.02 cd/m²). Asterisks show the points where thecurves for grafted and sham-operated eyes are statistically different(t-test, p<0.05).

FIG. 12 depicts in vitro maturation and degree of pigmentation indifferent batches of human ES cell-derived RPE cells. hES cells werematured to yield (A) light (L1), (B) medium (L2), and (C) heavy (L3)pigmentation levels. (A): Phase contrast image; scale bar 200 gm. (B andC): Hoffman modulation contrast image; scale bar=100 μm.

FIG. 13 depicts comparative assessment of hES cell-RPE cells usingreal-time polymerase chain reaction (PCR) and Western blot analyses.(A): Reverse transcription-PCR analysis of genes specific to hES cells,neuroectoderm, and terminally differentiated RPE cells examinedthroughout the in vitro differentiation process. Time points correspondto hES cells, EBs, plated EBs representing early intermediates (EB/RPE),a mixed population of cells containing newly differentiated RPE cells,remaining progenitors (Mixed), purified RPE (corresponding to FIG. 12A),and fully-mature RPE (corresponding to FIG. 12C). (B): Western blotanalysis of hESC-specific and RPE-specific markers. APRE-19 cells (toplane) show an inconclusive pattern of proteomic marker expression. Actinis used as protein loading control. RPE (bottom lane) derived from hEScells (middle lane) do not express the hES cell-specific proteins Oct-4,NANOG, Rex-1, TDGF1, and DPPA4. However, RPE cells express RPE65,CRALBP, PEDF, Bestrophin, PAX6, Pax2, Otx2, MitF, and Tyr—all markers ofdifferentiated RPE.

FIG. 14 depicts principal components analysis plot. Component 1represents 69% of the variability represents the cell type, whereascomponent 2 represents the cell line (i.e., genetic variability). Anear-linear scatter of gene expression profiles characterizes thedevelopmental ontogeny of RPE derived from hES cells.

FIG. 15 depicts (A): visual acuity as measured by the optomotor responseshows that animals treated with 5,000, 20,000, 50,000, 75,000, and100,000 cells performed significantly better than those with shaminjection and untreated controls (p<0.01) at P90 days (e.g., a figure of0.563 c/d compared with 0.6 c/d in normal rat). (B): Visual acuitytested in Elovl4 mice at several time points after subretinal injectionof human RPE cells showed that cell-injected animals performedsignificantly better than medium-injected and untreated controls(p<0.05). Some showed a figure of 0.32 c/d at P63 compared with 0.35 c/din normal mice, whereas control animals had a figure of 0.28 c/d. (C-F):Luminance threshold responses recorded across the superior colliculus(SC); each curve (average±SEM) shows the percent of retinal area(y-axis) where the visual threshold is less than the corresponding valueat x-axis (log units, relative to background illumination 0.02 cd/m²).Cell-injected groups are significantly better than controls: the curvesshowed that 28% of the area in the SC in animals with the (C) 20,000 RPEcell dose; (D) about 45% with the 50,000 RPE cell dose; (E) about 40%with the 75,000 RPE cell dose; (F) about 60% with the 100,000 RPE celldose; and only 3% in medium control had thresholds of 2.2 log units.Dashed lines—cell-treated and Solid lines—medium control. Abbreviation:c/d, cycles/degree.

FIG. 16 depicts changes in acuity and luminance threshold with time.Batch and longevity of effect as measured by visual acuity:cell-injected groups at all the time points (P60-P240) had significantlyhigher visual acuities than controls (p<0.01); however, there is nosubstantial difference with different pigment levels (p>0.05).Abbreviation: c/d, cycles/degree.

FIG. 17 depicts a comparison of the effects of pigmentation on theefficacy of RPE cells in a RCS rat model. The rats were transplantedwith 50,000 RPE cells with low, medium, or high pigmentation levels.These rats were compared to sham surgery and untreated controls.

FIG. 18 two examples of luminance threshold maps from mice receiving a100,000 RPE cell dose with medium pigmentation. The luminance thresholdsshow serious deterioration on the untreated side, with more than onehalf the area being nonresponsive at P187 compared with P98, whereasresponsiveness is still sensitive on the cell-injected side, althoughsome reduction in thresholds has occurred (0.7 log units at P98 vs. 1.0log units at P187).

FIG. 19 depicts histological examination of cell-injected and untreatedRCS retinas, showing photoreceptors in (A) normal, (B) cell injected,and (C) untreated eyes at P90 (arrows in B point to rescuedphotoreceptors; arrows in C indicate remaining photoreceptors). (D-F):Photoreceptors rescued at (D) 5,000 and (E and F) 50,000 dose (arrows inE indicate rescued photoreceptors; cone arrestin showed rescued conephotoreceptors in F). (G): Immunofluorescence- and (H)immunohistochemical-stained human specific antibody showing donor cells(arrows) formed a layer closely contact with the host RPE layer at P240.(I): Typical untreated retina at P240 with disorganized retinallamination (left arrow indicates RPE cells migrating into inner retina;right arrow indicates disrupted inner nuclear layer). Scale bars 25 μm.Abbreviations: INL: inner nuclear layer; IPL: inner plexiform layer;ONL: outer nuclear layer; RPE, retinal pigment epithelium; RGC: retinalganglion cells.

DETAILED DESCRIPTION OF THE INVENTION

in order that the invention herein described may be fully understood,the following detailed description is set forth. Various embodiments ofthe invention are described in detail and may be further illustrated bythe provided examples.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe invention or testing of the present invention, suitable methods andmaterials are described below. The materials, methods and examples areillustrative only, and are not intended to be limiting.

In order to further define the invention, the following terms anddefinitions are provided herein.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise.

“Effective amount,” as used herein, refers broadly to the amount of acompound or cells that, when administered to a patient for treating adisease, is sufficient to effect such treatment for the disease. Theeffective amount may be an amount effective for prophylaxis, and/or anamount effective for prevention. The effective amount may be an amounteffective to reduce, an amount effective to prevent the incidence ofsigns/symptoms, to reduce the severity of the incidence ofsigns/symptoms, to eliminate the incidence of signs/symptoms, to slowthe development of the incidence of signs/symptoms, to prevent thedevelopment of the incidence of signs/symptoms, and/or effectprophylaxis of the incidence of signs/symptoms. The “effective amount”may vary depending on the disease and its severity and the age, weight,medical history, susceptibility, and preexisting conditions, of thepatient to be treated. The term “effective amount” is synonymous with“therapeutically effective amount” for purposes of this invention.

“Embryo” or “embryonic,” as used herein refers broadly to a developingcell mass that has not implanted into the uterine membrane of a maternalhost. An “embryonic cell” is a cell isolated from or contained in anembryo. This also includes blastomeres, obtained as early as thetwo-cell stage, and aggregated blastomeres.

“Embryonic stem cells” (ES cells), as used herein, refers broadly tocells derived from the inner cell mass of blastocysts or morulae thathave been serially passaged as cell lines. The ES cells may be derivedfrom fertilization of an egg cell with sperm or DNA, nuclear transfer,parthenogenesis, or by means to generate ES cells with homozygosity inthe HLA region. ES cells may also refer to cells derived from a zygote,blastomeres, or blastocyst-staged mammalian embryo produced by thefusion of a sperm and egg cell, nuclear transfer, parthenogenesis, orthe reprogramming of chromatin and subsequent incorporation of thereprogrammed chromatin into a plasma membrane to produce a cell.Embryonic stem cells, regardless of their source or the particularmethod used to produce them, can be identified based on the: (i) abilityto differentiate into cells of all three germ layers, (ii) expression ofat least Oct-4 and alkaline phosphatase, and (iii) ability to produceteratomas when transplanted into immunocompromised animals.

“Embryo-derived cells” (EDC), as used herein, refers broadly tomorula-derived cells, blastocyst-derived cells including those of theinner cell mass, embryonic shield, or epiblast, or other pluripotentstem cells of the early embryo, including primitive endoderm, ectoderm,and mesoderm and their derivatives. “EDC” also including blastomeres andcell masses from aggregated single blastomeres or embryos from varyingstages of development, but excludes human embryonic stem cells that havebeen passaged as cell lines.

“Macular degeneration,” as used herein, refers broadly to diseasescharacterized by a progressive loss of central vision associated withabnormalities of Bruch's membrane, the neural retina, and the retinalpigment epithelium. Macular degeneration diseases include but are notlimited to age-related macular degeneration, North Carolina maculardystrophy, Sorsby's fundus dystrophy, Stargardt's disease, patterndystrophy, Best disease, malattia leventinese, Doyne's honeycombchoroiditis, dominant drusen, and radial drusen.

“Pluripotent stem cell,” as used herein, refers broadly to a cellcapable of prolonged or virtually indefinite proliferation in vitrowhile retaining their undifferentiated state, exhibiting normalkaryotype (e.g., chromosomes), and having the capacity to differentiateinto all three germ layers (i.e., ectoderm, mesoderm and endoderm) underthe appropriate conditions.

“Pluripotent embryonic stem cells,” as used herein, refers broadly cellsthat: (a) are capable of inducing teratomas when transplanted inimmunodeficient (SCID) mice; (b) are capable of differentiating to celltypes of all three germ layers (e.g., ectodermal, mesodermal, andendodermal cell types); and (c) express at least one molecular embryonicstem cell markers (e.g., express Oct 4, alkaline phosphatase, SSEA-3surface antigen, SSEA-4 surface antigen, NANOG, TRA-1-60, TRA-1-81,SOX2, REX1).

“RPE cell,” “differentiated RPE cell,” “ES-derived RPE cell,” and asused herein, may be used interchangeably throughout to refer broadly toan RPE cell differentiated from a pluripotent stem cell using a methodof the invention. The term is used generically to refer todifferentiated RPE cells, regardless of the level of maturity of thecells, and thus may encompass RPE cells of various levels of maturity.RPE cells can be visually recognized by their cobblestone morphology andthe initial appearance of pigment. RPE cells can also be identifiedmolecularly based on substantial lack of expression of embryonic stemcell markers such as Oct-4 and NANOG, as well as based on the expressionof RPE markers such as RPE-65, PEDF, CRALBP, and bestrophin. Thus,unless otherwise specified, RPE cells, as used herein, refers to RPEcells differentiated in vitro from pluripotent stem cells.

“Mature RPE cell” and “mature differentiated RPE cell,” as used herein,may be used interchangeably throughout to refer broadly to changes thatoccur following initial differentiating of RPE cells. Specifically,although RPE cells can be recognized, in part, based on initialappearance of pigment, after differentiation mature RPE cells can berecognized based on enhanced pigmentation.

“Pigmented,” as used herein refers broadly to any level of pigmentation,for example, the pigmentation that initial occurs when RPE cellsdifferentiate from ES cells. Pigmentation may vary with cell density andthe maturity of the differentiated RPE cells. The pigmentation of a RPEcell may be the same as an average RPE cell after terminaldifferentiation of the RPE cell. The pigmentation of a RPE cell may bemore pigmented than the average RPE cell after terminal differentiationof the RPE cell. The pigmentation of a RPE cell may be less pigmentedthan the average RPE cell after terminal differentiation.

“Signs” of disease, as used herein, refers broadly to any abnormalityindicative of disease, discoverable on examination of the patient; anobjective indication of disease, in contrast to a symptom, which is asubjective indication of disease.

“Symptoms” of disease as used herein, refers broadly to any morbidphenomenon or departure from the normal in structure, function, orsensation, experienced by the patient and indicative of disease.

“Therapy,” “therapeutic,” “treating,” or “treatment”, as used herein,refers broadly to treating a disease, arresting or reducing thedevelopment of the disease or its clinical symptoms, and/or relievingthe disease, causing regression of the disease or its clinical symptoms.Therapy encompasses prophylaxis, prevention, treatment, cure, remedy,reduction, alleviation, and/or providing relief from a disease, signs,and/or symptoms of a disease. Therapy encompasses an alleviation ofsigns and/or symptoms in patients with ongoing disease signs and/orsymptoms (e.g., blindness, retinal deterioration.) Therapy alsoencompasses “prophylaxis” and “prevention”. Prophylaxis includespreventing disease occurring subsequent to treatment of a disease in apatient or reducing the incidence or severity of the disease in apatient. The term “reduced”, for purpose of therapy, refers broadly tothe clinical significant reduction in signs and/or symptoms. Therapyincludes treating relapses or recurrent signs and/or symptoms (e.g.,retinal degeneration, loss of vision.) Therapy encompasses but is notlimited to precluding the appearance of signs and/or symptoms anytime aswell as reducing existing signs and/or symptoms and eliminating existingsigns and/or symptoms. Therapy includes treating chronic disease(“maintenance”) and acute disease. For example, treatment includestreating or preventing relapses or the recurrence of signs and/orsymptoms (e.g., blindness, retinal degeneration).

Retinal Pigment Epithelium (RPE) Cells

The present invention provides RPE cells that may be differentiated frompluripotent stem cells, such as human embryonic stem cells, and aremolecularly distinct from embryonic stem cells, adult-derived RPE cells,and fetal-derived RPE cells. The inventors surprisingly discovered thatthe method by which the RPE cells are produced from a pluripotent stemcell is a critical factor in determining the structural and functionalcharacteristics of the resulting RPE cells. The inventors found that theRPE cells produced by the methods described produced a different RPEcell product than previous methods and sources of RPE cells. Forexample, the manufacturing process steps described herein impartdistinctive structural and functional characteristics to the final RPEcell product such that these cells closely resemble native RPE cells andare distinct from fetal derived RPE cells or RPE cell lines (e.g.,APRE19). Further, the methods of producing RPE cells described hereinare not permissive to ES cells. Thus, as ES cells cannot persist in theculture processes described herein, and they do not pose an unacceptablerisk of contamination in the RPE cell cultures and preparations.

The cell types provided by this invention include, but are not limitedto, RPE cells, RPE progenitor cells, iris pigmented epithelial (IPE)cells, and other vision associated neural cells, such as internuncialneurons (e.g., “relay” neurons of the inner nuclear layer (INL)) andamacrine cells. The invention also provides retinal cells, rods, cones,and corneal cells as well as cells providing the vasculature of the eye.

The RPE cells may be used for treating retinal degeneration diseases dueto retinal detachment, retinal dysplasia, or retinal atrophy orassociated with a number of vision-altering ailments that result inphotoreceptor damage and blindness, such as, choroideremia, diabeticretinopathy, macular degeneration (e.g., age-related maculardegeneration), retinitis pigmentosa, and Stargardt's Disease (fundusflavimaculatus).

The RPE cells may be stable, terminally differentiated RPE cells that donot de-differentiate to a non-RPE cell type. The RPE cells describedherein may be functional RPE cells, characterized by the ability tointegrate into the retina upon corneal, sub-retinal, or otheradministration into an animal.

In order to characterize developmental stages during the embryonic stemcell (ES) differentiation process into retinal pigmented epithelium(RPE), several assays were used to identify the expression levels ofgenes key to each representative stage of development. It was discoveredthat several genes were expressed at the mRNA and protein levels in RPEcells. The expression level of ES and RPE cell markers may be done atthe mRNA by, for example, PCR (e.g., RT-PCT, quantitative PCR, real-timePCR) or Northern blotting, or at the protein level by, for example,Western blot, immunoblot, or other immunoassays.

The pluripotency of embryonic stem cells is maintained in part by thedelicate reciprocal balance of the two transcription factors Oct4(Pou5f1) and NANOG. During ES cell differentiation, the expression ofthese genes is downregulated, and recent evidence has suggestedhypermethylation of the genes encoding these proteins to be responsible.Loss of the expression of either or both of these genes results intranscriptional activation of genes associated with cellulardifferentiation. For instance, it was discovered that PAX6 acts withPAX2 to terminally differentiate mature RPE cells via coordination ofMit-F and Otx2 to transcribe RPE-specific genes such as Tyrosinase(Tyr), and downstream targets such as RPE-65, Bestrophin, CRALBP, andPEDF.

The RPE cells may express RPE cell markers listed in Table 5. Forexample, the expression level of the RPE cell genes RPE65, PAX2, PAX6,and tyrosinase, bestrophin, PEDF, CRALBP, Otx2, and MitF may beequivalent to that in naturally occurring RPE cells. The level ofmaturity of the RPE cells may assessed by expression of at least one ofPAX2, PAX6, and tyrosinase, or their respective expression levels.

In contrast, the RPE cells may not express. ES cell markers listed inTable 6. For example, the expression levels of the ES cell genes Oct-4,NANOG, and/or Rex-1 may be about 100-1000 fold lower in RPE cells thanin ES cells. For example, the RPE cells may substantially lackexpression of ES cell markers including but not limited to Octamerbinding protein 4 (Oct-4, a.k.a., Pou5f1), stage specific embryonicantigens (SSEA)-3 and SSEA-4, tumor rejection antigen (TRA)-1-60,TRA-1-80, alkaline phosphatase, NANOG, and Rex-1. Thus, in comparison toES cells, RPE cells are substantially lack expression of Oct-4, NANOG,and/or Rex-1.

The RPE cells described herein may also show elevated expression levelsof alpha integrin subunits 1-6 or 9 as compared to uncultured RPE cellsor other RPE cell preparations. The RPE cells described herein may alsoshow elevated expression levels of alpha integrin subunits 1, 2, 3, 4,5, or 9. The RPE cells described herein may be cultured under conditionsthat promote the expression of alpha integrin subunits 1-6. For example,the RPE cells may be cultured with integrin-activating agents includingbut not limited to manganese and the activating monoclonal antibody(mAb) TS2/16. See Afshari, et al. Brain (2010) 133(2): 448-464. The RPEcells may be plated on laminin (1 μg/mL) and exposed to Mn²⁺ (500 μM)for at least about 8, 12, 24, 36, or 48 hours. Also, the RPE cells maybe cultured for several passages (e.g., at least about 4, 5, 6, 7, or 8passages) which increases alpha integrin subunit expression.

Table 1 describes some characteristics of the RPE cells that may be usedto identify or characterize the RPE cells. In particular, the RPE cellsmay exhibit a normal karyotype, express RPE markers, and not express hESmarkers.

TABLE 1 Parameters of RPE cells Specification for RepresentativeParameter Lot of RPE Cells Karyotype 46, XX Normal Morphology at harvestNormal cellular morphology, medium pigmentation Post-thaw Viable CellCount >70% qPCR Testing— Present Presence of RPE Markers BestrophinRPE-65 CRALBP PEDF PAX6 MITF qPCR Testing— Absent Absence of hES MarkersOct-4 NANOG Rex-1 Sox2 Immunostaining— Present Presence of RPE MarkersBestrophin CRALBP PAX6 MITF ZO-1 Immunostaining— Absent Absence of hESmarkers Oct-4 Alkaline Phosphatase

The distinct expression pattern of mRNA and proteins in the RPE cells ofthe invention constitutes a set of markers that separate these RPE cellsfrom cells in the art, such as hES cells, ARPE-19 cells, and fetal RPEcells. Specifically, these cells are different in that they can beidentified or characterized based on the expression or lack ofexpression, which may be assessed by mRNA or protein level, of at leastone marker. For example, the RPE cells may be identified orcharacterized based on expression or lack of expression of at least onemarker listed in Tables 5 or 6. See also Liao, et al. (2010) HumanMolecular Genetics 19(21): 4229-38. The RPE cells may also be identifiedand characterized, as well as distinguished from other cells, based ontheir structural properties. Thus, the RPE cells described hereinexpressed multiple genes that were not expressed in hES cells, fetal RPEcells, or ARPE-19 cells. See WO 2009/051671; See also Dunn, et al.(1996) Exp Eye Res. 62(2): 155-169.

The RPE cells described herein may also be identified and characterizedbased on the degree of pigmentation of the cell. Pigmentationpost-differentiation is not indicative of a change in the RPE state ofthe cells (e.g., the cells are still differentiated RPE cells). Rather,the changes in pigment post-differentiation correspond to the density atwhich the RPE cells are cultured and maintained. Mature RPE cells haveincreased pigmentation that accumulates after initial differentiation.For example, the RPE cells described herein may be mature RPE cells withincreased pigmentation in comparison to differentiated RPE cells.Differentiated RPE cells that are rapidly dividing are lightlypigmented. However, when cell density reaches maximal capacity, or whenRPE cells are specifically matured, RPE take on their characteristicphenotypic hexagonal shape and increase pigmentation level byaccumulating melanin and lipofuscin. As such, initial accumulation ofpigmentation serves as an indicator of RPE differentiation and increasedpigmentation associated with cell density serves as an indicator of RPEmaturity. For example, the RPE cells may be pigmented, to at least someextent. For example, the RPE cell may be derived from a human embryonicstem cell, which cell is pigmented and expresses at least one gene thatis not expressed in a cell that is not a human retinal pigmentedepithelial cell.

Mature RPE cells can be subcultured at a lower density, such that thepigmentation decreases. In this context, mature RPE cells may becultured to produce RPE cells. Such RPE cells are still differentiatedRPE cells that express markers of RPE differentiation. Thus, in contrastto the initial appearance of pigmentation that occurs when RPE cellsbegin to differentiate, pigmentation changes post-differentiation arephenomenological and do not reflect dedifferentiation of the cells awayfrom an RPE fate.

The RPE cells described herein may maintain their phenotype for a longperiod of time in vitro. For example, the RPE cells may maintain theirphenotype for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 passages. The RPE cells may maintain theirphenotype for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 days. The RPE cells may maintain theirphenotype for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.

Moreover, the RPE cells described herein may maintain their phenotypefollowing transplantation. The RPE cells may maintain their phenotypefor the lifespan of the receipt after transplantation. For example, theRPE cells may maintain their phenotype following transplantation for atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or days. Further, the RPE cells may maintain their phenotypefollowing transplantation for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 weeks. The RPE cells may maintain their phenotype followingtransplantation for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12 months. The RPE cells may maintain their phenotype followingtransplantation for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 years.

The RPE cells have an increased ability to prevent neovascularization.The RPE cells may be produced by aging a somatic cell from a patientsuch that telomerase is shortened where at least 10% of the normalreplicative lifespan of the cell has been passed, then the use of saidsomatic cell as a nuclear transfer donor cell to create cells thatoverexpress angiogenesis inhibitors such as Pigment Epithelium DerivedFactor (PEDF/EPC-1). Alternatively such cells may be geneticallymodified with exogenous genes that inhibit neovascularization.

Preparations of RPE Cells

The present invention provides preparations of RPE cells. The inventiondescribed herein provides RPE cells, substantially purified populationsof RPE cells, pharmaceutical preparations comprising RPE cells, andcryopreserved preparations of the RPE cells. The RPE cells describedherein may be substantially free of at least one protein, molecule, orother impurity that is found in its natural environment (e.g.,“isolated”.) The RPE cells may be mammalian, including, human RPE cells.The invention also provides human RPE cells, a substantially purifiedpopulation of human RPE cells, pharmaceutical preparations comprisinghuman RPE cells, and cryopreserved preparations of the human RPE cells.The preparation may be a preparation comprising human embryonic stemcell-derived RPE cells, human iPS cell-derived RPE cells, andsubstantially purified (with respect to non-RPE cells) preparationscomprising differentiated ES-derived RPE cells.

The RPE cell populations may include differentiated RPE cells of varyinglevels of maturity, or may be substantially pure with respect todifferentiated RPE cells of a particular level of maturity. The RPEcells may be a substantially purified preparation comprising RPE cellsof varying levels of maturity/pigmentation. For example, thesubstantially purified culture of RPE cells may contain bothdifferentiated RPE cells and mature differentiated RPE cells. Amongstthe mature RPE cells, the level of pigment may vary. However, the matureRPE cells may be distinguished visually from the RPE cells based on theincreased level of pigmentation and the more columnar shape. Thesubstantially purified preparation of RPE cells comprises RPE cells ofdiffering levels of maturity (e.g., differentiated RPE cells and maturedifferentiated RPE cells). In such instances, there may be variabilityacross the preparation with respect to expression of markers indicativeof pigmentation. The pigmentation of the RPE cells in the cell culturemay be homogeneous. Further, the pigmentation of the RPE cells in thecell culture may be heterogeneous, and the culture of RPE cells maycomprise both differentiated RPE cells and mature RPE cells.Preparations comprising RPE cells include preparations that aresubstantially pure, with respect to non-RPE cell types, but whichcontain a mixture of differentiated RPE cells and mature differentiatedRPE cells. Preparations comprising RPE cells also include preparationsthat are substantially pure both respect to non-RPE cell types and withrespect to RPE cells of other levels of maturity.

The percentage of mature differentiated RPE cells in the culture may bereduced by decreasing the density of the culture. Thus, the methodsdescribed herein may further comprise subculturing a population ofmature RPE cells to produce a culture containing a smaller percentage ofmature RPE cells. The number of RPE cells in the preparation includesdifferentiated RPE cells, regardless of level of maturity and regardlessof the relative percentages of differentiated RPE cells and maturedifferentiated RPE cells. The number of RPE cells in the preparationrefers to the number of either differentiated RPE cells or mature RPEcells. The preparation may comprise at least about 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% differentiated RPEcells. The preparation may comprise at least about 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% mature RPE cells.The RPE cell preparation may comprise a mixed population ofdifferentiated RPE cells and mature RPE cells.

The invention provides a cell culture comprising human RPE cells whichare pigmented and express at least one gene that is not expressed in acell that is not a human RPE. For example, although such RPE cells mayhave substantially the same expression of RPE65, PEDF, CRALBP, andbestrophin as a natural human RPE cell. The RPE cells may vary,depending on level of maturity, with respect to expression of one ormore of PAX2, Pax-6, MitF, and/or tyrosinase. Note that changes inpigmentation post-differentiation also correlate with changes in PAX2expression. Mature RPE cells may be distinguished from RPE cells by thelevel of pigmentation, level of expression of PAX2, Pax-6, and/ortyrosinase. For example, mature RPE cells may have a higher level ofpigmentation or a higher level of expression of PAX2, Pax-6, and/ortyrosinase compared to RPE cells.

The preparations may be substantially purified, with respect to non-RPEcells, comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% RPE cells. The RPE cell preparation maybe essentially free of non-RPE cells or consist of RPE cells. Forexample, the substantially purified preparation of RPE cells maycomprise less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, or 1% non-RPE cell type. For example, the RPE cell preparation maycomprise less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%,0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%,0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%,0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or0.0001% non-RPE cells.

The RPE cell preparations may be substantially pure, both with respectto non-RPE cells and with respect to RPE cells of other levels ofmaturity. The preparations may be substantially purified, with respectto non-RPE cells, and enriched for mature RPE cells. For example, in RPEcell preparations enriched for mature RPE cells, at least about 30%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% of the RPE cells are matureRPE cells. The preparations may be substantially purified, with respectto non-RPE cells, and enriched for differentiated RPE cells rather thanmature RPE cells. For example, at least about 30%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% of the RPE cells may be differentiated RPE cellsrather than mature RPE cells.

The RPE cell preparations may comprise at least about 1×10³, 2×10³,3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴,4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵,5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶,6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷,7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸,8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹,9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰,or 9×10¹⁰ RPE cells. The RPE cell preparations may comprise at leastabout 5,000-10,000, 50,000-100,000, 100,000-200,000, 200,000-500,000,300,000-500,000, or 400,000-500,000 RPE cells. The RPE cell preparationmay comprise at least about 20,000-50,000 RPE cells. Also, the RPE cellpreparation may comprise at least about 5,000, 10,000, 20,000, 30,000,40,000, 50,000, 60,000, 70,000, 75,000, 80,000, 100,000, or 500,000 RPEcells.

The RPE cell preparations may comprise at least about 1×10³, 2×10³,3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴,4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵,5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶,6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10′, 5×10⁷, 6×10⁷,7×10′, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸,8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹,9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰,or 9×10¹⁰ RPE cells/mL. The RPE cell preparations may comprise at leastabout 5,000-10,000, 50,000-100,000, 100,000-200,000, 200,000-500,000,300,000-500,000, or 400,000-500,000 RPE cells/mL. The RPE cellpreparation may comprise at least about 20,000-50,000 RPE cells/mL.Also, the RPE cell preparation may comprise at least about 5,000,10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, 100,000,or 500,000 RPE cells/mL.

The preparations described herein may be substantially free ofbacterial, viral, or fungal contamination or infection, including butnot limited to the presence of HIV-1, HIV-2, HBV, HCV, CMV, HTLV-1,HTLV-2, parvovirus B19, Epstein-Barr virus, or herpesvirus 6. Thepreparations described herein may be substantially free of mycoplasmacontamination or infection.

The RPE cells described herein may also act as functional RPE cellsafter transplantation where the RPE cells form a monolayer between theneurosensory retina and the choroid in the patient receiving thetransplanted cells. The RPE cells may also supply nutrients to adjacentphotoreceptors and dispose of shed photoreceptor outer segments byphagocytosis. Additionally, the RPE cells described herein may haveundergone less senescence than cells derived from eye donors (e.g., theRPE cells are “younger” than those of eye donors). This allows the RPEcell described herein to have a longer useful lifespan than cellsderived from eye donors.

The preparations comprising RPE cells may be prepared in accordance withGood Manufacturing Practices (GMP) (e.g., the preparations areGMP-compliant) and/or current Good Tissue Practices (GTP) (e.g., thepreparations may be GTP-compliant.)

RPE Cell Cultures

The present invention also provides substantially purified cultures ofRPE cells, including human RPE cells. The RPE cultures described hereinmay comprise at least about 1,000; 2,000; 3,000; 4,000; 5,000; 6,000;7,000; 8,000; or 9,000 RPE cells. The culture may comprise at leastabout 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴,1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶,2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷,3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸,4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹,5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰,5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, or 9×10 RPE cells.

The RPE cells are further cultured to produce a culture of mature RPEcells. The RPE cells may be matured, and the RPE cells may be furthercultured in, for example MDBK-MM medium until the desired level ofmaturation is obtained. This may be determined by monitoring theincrease in pigmentation level during maturation. As an alternative toMDBK-MM medium, a functionally equivalent or similar medium, may beused. Regardless of the particular medium used to mature the RPE cells,the medium may optionally be supplemented with a growth factor or agent.Both RPE cells and mature RPE cells are differentiated RPE cells.However, mature RPE cells are characterized by increased level ofpigment in comparison to differentiated RPE cells. The level of maturityand pigmentation may be modulated by increasing or decreasing thedensity of the culture of differentiated RPE cells. Thus, a culture ofRPE cells may be further cultured to produce mature RPE cells.Alternatively, the density of a culture containing mature RPE cells maybe decreased to decrease the percentage of mature differentiated RPEcells and increase the percentage of differentiated RPE cells.

The RPE cells may be identified by comparing the messenger RNAtranscripts of such cells with cells derived in vivo. An aliquot ofcells is taken at various intervals during the differentiation ofembryonic stem cells to RPE cells and assayed for the expression of anyof the markers described above. These characteristic distinguishdifferentiated RPE cells.

The RPE cell culture may be a substantially purified culture comprisingat least about 30%, 35%, 40%, or 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%differentiated RPE cells. The substantially purified culture maycomprise at least about 30%, 35%, 40%, or 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%mature differentiated RPE cells.

The RPE cell cultures may be prepared in accordance with GoodManufacturing Practices (GMP) (e.g., the cultures are GMP-compliant)and/or current Good Tissue Practices (GTP) (e.g., the cultures may beGTP-compliant.)

Cryopreserved Preparations of RPE Cells

RPE cells may be frozen for storage. The RPE cells may be stored by anyappropriate method known in the art (e.g., cryogenically frozen) and maybe frozen at any temperature appropriate for storage of the cells. Forexample, the cells may be frozen at about −20° C., −80° C., −120° C.,−130° C., −135° C., −140° C., −150° C., −160° C., −170° C., −180° C.,−190° C., −196° C., at any other temperature appropriate for storage ofcells. Cryogenically frozen cells may be stored in appropriatecontainers and prepared for storage to reduce risk of cell damage andmaximize the likelihood that the cells will survive thawing. RPE cellsmay be cryopreserved immediately following differentiation, following invitro maturation, or after some period of time in culture. The RPE cellsmay also be maintained at room temperature, or refrigerated at, forexample, about 4° C.

Similarly provided are methods of cryopreserving RPE cells. The RPEcells may be harvested, washed in buffer or media, counted, concentrated(via centrifugation), formulated in freezing media (e.g., 90% FBS/10%DMSO), or any combination of these steps. For example, the RPE cells maybe seeded in several culture vessels and serially expanded. As the RPEcells are harvested and maintained in FBS at about 4° C. while severalflasks of RPE cells are combined into a single lot. The RPE cells may bealso washed with saline solution (e.g., DPBS) at least 1, 2, 3, 4, or 5times. Further, the RPE cells may be cryopreserved after dystrophin isorganized at the cell membrane and PAX6 expression is low. In addition,the vials may be labeled, with a primary and/or secondary label. Theinformation on the label may include the type of cell (e.g., hRPEcells), the lot number and date, the number of cells (e.g., 1×10⁶cells/mL), the expiration date (e.g., recommended date by which the vialshould be used), manufacture information (e.g., name and address),warnings, and the storage means (e.g., storage in liquid nitrogen).

Cryopreserved RPE cell preparations described herein may comprise atleast about 50,000-100,000 RPE cells. The cryopreserved RPE cellpreparations may also comprise at least about 20,000-500,000 RPE cells.Also, the cryopreserved RPE cell preparations may comprise at leastabout 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000,80,000, or 100,000 RPE cells. The cryopreserved RPE cell preparationsmay comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 10,000,20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, 100,000, or500,000 RPE cells. The cryopreserved RPE cell preparations may compriseat least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000,9,000, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6)(10⁴, 7×10⁴, 8×10⁴, 9×10⁴,1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶,2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷,3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸,4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹,5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, or 9×10⁹RPE cells. The RPE cells of thecryopreserved RPE cell preparations may be mammalian RPE cells,including human RPE cells.

Further, the cryopreserved RPE cell preparations described herein maycomprise at least about 50,000-100,000 RPE cells/mL. The cryopreservedRPE cell preparations may also comprise at least about 20,000-500,000RPE cells/mL. Also, the cryopreserved RPE cell preparations may compriseat least about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000,75,000, 80,000, and 100,000 RPE cells/mL. The cryopreserved RPE cellpreparations may comprise at least about 1,000, 2,000, 3,000, 4,000,5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000,100,000, or 500,000 RPE cells/mL. The cryopreserved RPE cellpreparations may comprise at least about 1,000, 2,000, 3,000, 4,000,5,000, 6,000, 7,000, 8,000, 9,000, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴,6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵,7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶,8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷,9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸,1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰,2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, or 9×10¹⁰ RPEcells/mL. The RPE cells of the cryopreserved RPE cell preparations maybe mammalian RPE cells, including human RPE cells.

The RPE cells of the invention may be recovered from storage followingcryopreservation. The RPE cells recovered from cryopreservation alsomaintain their viability and differentiation status. For example, atleast about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of theRPE cells may retain viability and differentiation followingcryopreservation. Further, the RPE cells of the invention may becryopreserved and maintain their viability after being stored for atleast about 1, 2, 3, 4, 5, 6, or 7 days. The RPE cells of the inventionmay also be cryopreserved and maintain their viability after beingstored for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12months. The RPE cells of the invention may be cryopreserved and maintaintheir viability after being stored for at least about 1, 2, 3, 4, 5, 6,or 7 years. For example, the RPE cells of the invention may becryopreserved for at least about 4 years and show at least about 80%viability. The cryopreservation preparation comprising RPE cells may besubstantially free of DMSO.

Methods of Producing RPE Cells

The present invention provides a method of producing RPE cells frompluripotent stem cells. The cell types that may be produced using thisinvention include, but are not limited to, RPE cells, RPE progenitorcells, iris pigmented epithelial (IPE) cells, and other visionassociated neural cells, such as internuncial neurons (e.g., “relay”neurons of the inner nuclear layer (INL)) and amacrine cells.Additionally, retinal cells, rods, cones, and corneal cells may beproduced. Cells providing the vasculature of the eye may also beproduced by the methods described herein.

Without being bound to a particular theory, the inventors found that themethods described herein may act through FGF, EGF, WNT4, TGF-beta,and/or oxidative stress to signal MAP-Kinase and potential C-Junterminal Kinase pathways to induce the expression of the Paired-box 6(PAX6) transcription factor. PAX6 acts synergistically with PAX2 toterminally differentiate mature RPE via the coordination of Mit-F andOtx2 to transcribe RPE-specific genes such as Tyrosinase (Tyr), anddownstream targets such as RPE-65, Bestrophin, CRALBP, and PEDF. See WO2009/051671, FIG. 1.

The RPE cells described herein may be differentiated from pluripotentstem cells, such as human embryonic stem cells, and are molecularlydistinct from embryonic stem cells, adult-derived RPE cells, andfetal-derived RPE cells. The inventors surprisingly discovered that themethod by which the RPE cells are produced from a pluripotent stem cellis a critical factor in determining the structural and functionalcharacteristics of the resulting RPE cells. The inventors found that theRPE cells produced by the methods described produced a different RPEcell product than previous methods and sources of RPE cells. Forexample, the manufacturing process steps described herein impartdistinctive structural and functional characteristics to the final RPEcell product such that these cells closely resemble native RPE cells andare distinct from fetal derived RPE cells or RPE cell lines (e.g.,APRE19). Further, the methods of producing RPE cells described hereinare not permissive to ES cells. Thus, as ES cells cannot persist in theculture processes described herein, and they do not pose an unacceptablerisk of contamination in the RPE cell cultures and preparations.

The invention provides a method for producing a RPE cell comprising: (a)providing pluripotent stem cells; (b) culturing the pluripotent stemcells as embryoid bodies in nutrient rich, low protein medium, whereinthe medium optionally comprises serum free B-27 supplement; (c)culturing the embryoid bodies as an adherent culture in nutrient rich,low protein medium, wherein the medium optionally comprises serum freeB-27 supplement; (d) culturing the adherent culture of cells of (c) innutrient rich, low protein medium, wherein the medium does not compriseserum free B-27 supplement; (e) culturing the cells of (d) in mediumcapable of supporting growth of high-density somatic cell culture,whereby RPE cells appear in the culture of cells; (f) contacting theculture of (e) with an enzyme; (g) selecting the RPE cells from theculture and transferring the RPE cells to a separate culture containingmedium supplemented with a growth factor to produce an enriched cultureof RPE cells; and (g) propagating the enriched culture of RPE cells toproduce a RPE cell. These method steps may be performed at least once toproduce a substantially purified culture of RPE cells. Further, thesemethod steps may be repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10times to produce more RPE cells.

Additionally, the invention also provides a method for producing amature retinal pigment epithelial (RPE) cell comprising: (a) providingpluripotent stem cells; (b) culturing the pluripotent stem cells asembryoid bodies in nutrient rich, low protein medium, wherein the mediumoptionally comprises serum free B-27 supplement; (c) culturing theembryoid bodies as an adherent culture in nutrient rich, low proteinmedium, wherein the medium optionally comprises serum free B-27supplement; (d) culturing the adherent culture of cells of step (c) innutrient rich, low protein medium, wherein the medium does not compriseserum free B-27 supplement; (e) culturing the cells of (d) in mediumcapable of supporting growth of high-density somatic cell culture,whereby RPE cells appear in the culture of cells; (f) contacting theculture of (e) with an enzyme; (g) selecting the RPE cells from theculture and transferring the RPE cells to a separate culture containingmedium supplemented with a growth factor to produce an enriched cultureof RPE cells; (h) propagating the enriched culture of RPE cells; and (i)culturing the enriched culture of RPE cells to produce a mature RPEcell. These method steps may be performed at least once to produce asubstantially purified culture of mature RPE cells. Further, thesemethod steps may be repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10times to produce more mature RPE cells.

For any of the articulated steps, the cells may be cultured for at leastabout 1-10 weeks. For example, the cells may be cultured for at leastabout 3-6 weeks. For any of the articulated steps, the cells may becultured for between about 1 days and 50 days, for example, for at leastabout 1-3, 3-4,7, 4-9, 7-10, 7-12, 8-11, 9-12, 7-14, 14-21, and 3-45days. The cells may be cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50 days. The cells may be cultured for about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,or 24 hours. For example, the cells may be cultured for 2-4 and 3-6hours. For each of the above articulated method steps, the cells may becultured for the same period of time at each step or for differingperiods of time at one or more of the steps. Additionally, any of theabove articulated method steps may be repeated to produce more RPE cells(e.g., scaled up to produce large numbers of RPE cells).

In the methods described herein, the RPE cells may begin todifferentiate from amongst cells in the adherent culture of EBs. RPEcells may be visually recognized based on their cobblestone morphologyand the initial appearance of pigmentation. As RPE cells continue todifferentiate, clusters of RPE cells may be observed. See FIG. 4.

Mechanical or enzymatic methods are used to select RPE cells fromamongst clusters of non-RPE cells in a culture of embryoid body, or tofacilitate sub-culture of adherent cells. Exemplary mechanical methodsinclude, but are not limited to, titration with a pipette or cuttingwith a pulled needle. Exemplary enzymatic methods include, but are notlimited to, any enzymes appropriate for disassociating cells (e.g.,trypsin (e.g., Trypsin/EDTA), collagenase (e.g., collagenase B,collagenase IV), dispase, papain, mixture of collagenase and dispase, amixture of collagenase and trypsin). A non-enzymatic solution is used todisassociate the cells, such as a high EDTA-containing solution e.g.,Hanks-based cell disassociation buffer.

The RPE cells differentiate from the embryoid bodies. Isolating RPEcells from the EBs allows for the expansion of the RPE cells in anenriched culture in vitro. For human cells, RPE cells may be obtainedfrom EBs grown for less than 90 days. Further, RPE cells may arise inhuman EBs grown for at least about 7-14 days, 14-28 days, 28-45 days, or45-90 days. The medium used to culture pluripotent stem cells, embryoidbodies, and RPE cells may be removed and/or replaced with the same ordifferent media at any interval. For example, the medium may be removedand/or replaced after at least about 0-7 days, 7-10 days, 10-14 days,14-28 days, or 28-90 days. Further, the medium may be replaced at leastdaily, every other day, or at least every 3 days.

To enrich for RPE cells and to establish substantially purified culturesof RPE cells, RPE cells are dissociated from each other and from non-RPEcells using mechanical and/or chemical methods. A suspension of RPEcells may then be transferred to fresh medium and a fresh culture vesselto provide an enriched population of RPE cells. See FIG. 5.

RPE cells may be selected from the dissociated cells and culturedseparately to produce a substantially purified culture of RPE cells. RPEcells are selected based on characteristics associated with RPE cells.For example, RPE cells can be recognized by cobblestone cellularmorphology and pigmentation. In addition, there are several knownmarkers of the RPE, including cellular retinaldehyde-binding protein(CRALBP), a cytoplasmic protein that is also found in apical microvilli;RPE65, a cytoplasmic protein involved in retinoid metabolism;bestrophin, the product of the Best vitelliform macular dystrophy gene(VMD2), and pigment epithelium derived factor (PEDF), a 48 kD secretedprotein with angiostatic properties. The messenger RNA transcripts ofthese markers may be assayed using PCR (e.g., RT-PCR) or Northern blots.Also, the protein levels of these markers may be assaying usingimmunoblot technology or Western blots.

The RPE cells may also be selected based on cell function, such as byphagocytosis of shed rod and cone outer segments, absorption of straylight, vitamin A metabolism, regeneration of retinoids, and tissuerepair. Evaluation may also be performed using behavioral tests,fluorescent angiography, histology, tight junctions conductivity, orevaluation using electron microscopy.

The enriched cultures of RPE cells may be cultured in appropriatemedium, for example, EGM-2 medium. This, or a functionally equivalent orsimilar medium, may be supplemented with a growth factor or agent (e.g.,bFGF, heparin, hydrocortisone, vascular endothelial growth factor,recombinant insulin-like growth factor, ascorbic acid, or humanepidermal growth factor). The RPE cells may be phenotypically stableover a long period of time in culture (e.g., >6 weeks).

Pluripotent Stem Cells

The methods described herein may use pluripotent stem cells to produceRPE cells. Suitable pluripotent stem cells include but are not limitedto embryonic stem cells, embryo-derived stem cells, and inducedpluripotent stem cells, regardless of the method by which thepluripotent stem cells are derived. Pluripotent stem cells may begenerated using, for example, by methods known in the art. Exemplarypluripotent stem cells include embryonic stem cells derived from theinner cell mass (ICM) of blastocyst stage embryos, as well as embryonicstem cells derived from one or more blastomeres of a cleavage stage ormorula stage embryo (optionally without destroying the remainder of theembryo). Such embryonic stem cells may be generated from embryonicmaterial produced by fertilization or by asexual means, includingsomatic cell nuclear transfer (SCNT), parthenogenesis, cellularreprogramming, and androgenesis. Further, suitable pluripotent stemcells include but are not limited to human embryonic stem cells, humanembryo-derived stem cells, and human induced pluripotent stem cells,regardless of the method by which the pluripotent stem cells arederived.

The pluripotent stem cells (e.g., hES cells) may be cultured as asuspension culture to produce embryoid bodies (EBs). The embryoid bodiesmay be cultured in suspension for about 7-14 days. However, in certainembodiments, the EBs may be cultured in suspension for fewer than 7 days(less than 7, 6, 5, 4, 3, 2, or less than 1 day) or greater than 14days. The EBs may be cultured in medium supplemented with B-27supplement.

After culturing the EBs in suspension culture, the EBs may betransferred to produce an adherent culture. For example, the EBs may beplated onto gelatin coated plates in medium. When cultured as anadherent culture, the EBs may be cultured in the same type of media aswhen grown in suspension. The media may not supplemented with B-27supplement when the cells are cultured as an adherent culture. Also, themedium is supplemented with B-27 initially (e.g., for less than or equalto about 7 days), but then subsequently cultured in the absence of B-27for the remainder of the period as an adherent culture. The EBs may becultured as an adherent culture for at least about 14-28. However, incertain embodiments, the EBs may be cultured as an adherent culture forfewer than about 14 days (less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or less than 1 day) or greater than about 28 days.

Human Embryonic Stem Cells

Human embryonic stem (hES) cells may be used as a pluripotent stem cellin the methods described herein. Human embryonic stem cells (hES) areprogeny of the inner cell mass (ICM) of a blastocyst and may remainpluripotent virtually indefinitely. The hES cells may be derived fromone or more blastomeres of an early cleavage stage embryo, optionallywithout destroying the embryo. The hES cells may be cultured in any wayknown in the art, such as in the presence or absence of feeder cells.For example, the hES cells may be cultured in MDBK-GM, hESC Medium,INVITROGEN® Stem Cell Media, OptiPro SFM, VP-SFM, EGM-2, or MDBK-MM. SeeStem Cell Information (Culture of Human Embryonic Stem Cells (hESC))[NIH website, 2010]. The hES cells may be used and maintained inaccordance with GMP standards.

When grown in culture on a feeder layer in defined conditions hES cellsmaintain a specific morphology, forming flat colonies comprised ofsmall, tightly packed cells with a high ratio of nucleus to cytoplasm,clear boundaries between the cells, and sharp, refractile colonyborders. hES cells express a set of molecular markers, such as Octamerbinding protein 4 (Oct-4, a.k.a., Pou5f1), stage specific embryonicantigens (SSEA)-3 and SSEA-4, tumor rejection antigen (TRA)-1-60,TRA-1-80, alkaline phosphatase, NANOG, and Rex-1. Similar to the cellsof the ICM that differentiate into predetermined lineages, hES cells inculture may be induced to differentiate. For example, hES cells may bedifferentiated into human RPE under the defined conditions describedherein.

Human ES cells may produced using any method known in the art. Forexample, the hES cells may be derived from blastocyst stage embryos thatwere the product of in vitro fertilization of egg and sperm.Alternatively, the hES cells may be derived from one or more blastomeresremoved from an early cleavage stage embryo, optionally, withoutdestroying the remainder of the embryo. The hES cells may be producedusing nuclear transfer. Also, cryopreserved hES cells may be used.

Human embryonic stem cells that may be used include, but are not limitedto, MA01, MA09, ACT-4, No, 3, H1, H7, H9, H14 and ACT30 embryonic stemcells. See also NIH Human Embryonic Stem Cell Registry. An exemplaryhuman embryonic stem cell line that may be used is MA09 cells. Theisolation and preparation of MA09 cells was previously described inKlimanskaya, et al. (2006) “Human Embryonic Stem Cell lines Derived fromSingle Blastomeres.” Nature 444: 481-485.

The hES cells may be initially co-cultivated with murine embryonicfeeder cells (MEF) cells. The MEF cells may be mitotically inactivatedby exposure to mitomycin C prior to seeding hES cells in co-culture, andthus the MEFs do not propagate in culture. See FIG. 1. Additionally, hEScell cultures are examined microscopically and colonies containingnon-hES cell morphology are picked and discarded using a stem cellcutting tool. See FIG. 2. After the point of harvest of the hES cellsfor seeding for embryoid body formation no additional MEF cells are usedin the process. See FIG. 3. The time between MEF removal and RPE cellsdescribed herein harvest may be a minimum of at least one, two, three,four, or five passages and at least about 5, 10, 20, 30, 40, 50, 60, 70,80, 90, or 100 days in MEF-free cell culture. The time between MEFremoval and harvesting the RPE cells may also be a minimum of at leastabout 3 passages and at least about 80-90 days in MEF-free cell culture.Due to the methods of production described herein, the RPE cell culturesand preparations described herein may be substantially free of mouseembryo fibroblasts (MEF) and human embryonic stem cells (hES).

Induced Pluripotent Stein Cells (iPS Cells)

Further exemplary pluripotent stem cells include induced pluripotentstem cells (iPS cells) generated by reprogramming a somatic cell byexpressing or inducing expression of a combination of factors(“reprogramming factors”). iPS cells may be generated using fetal,postnatal, newborn, juvenile, or adult somatic cells. iPS cells may beobtained from a cell bank. Alternatively, iPS cells may be newlygenerated by methods known in the art prior to commencingdifferentiation to RPE cells. The making of iPS cells may be an initialstep in the production of RPE cells. iPS cells may be specificallygenerated using material from a particular patient or matched donor withthe goal of generating tissue-matched RPE cells. iPS cells are universaldonor cells that are not substantially immunogenic.

The induced pluripotent stem cell may be produced by expressing orinducing the expression of one or more reprogramming factors in asomatic cell. The somatic cell is a fibroblast, such as a dermalfibroblast, synovial fibroblast, or lung fibroblast, or anon-fibroblastic somatic cell. The somatic cell is reprogrammed byexpressing at least 1, 2, 3, 4, 5. The reprogramming factors may beselected from Oct 3/4, Sox2, NANOG, Lin28, c-Myc, and Klf4. Expressionof the reprogramming factors may be induced by contacting the somaticcells with at least one agent, such as a small organic molecule agents,that induce expression of reprogramming factors.

The somatic cell may also be reprogrammed using a combinatorial approachwherein the reprogramming factor is expressed (e.g., using a viralvector, plasmid, and the like) and the expression of the reprogrammingfactor is induced (e.g., using a small organic molecule.) For example,reprogramming factors may be expressed in the somatic cell by infectionusing a viral vector, such as a retroviral vector or a lentiviralvector. Also, reprogramming factors may be expressed in the somatic cellusing a non-integrative vector, such as an episomal plasmid. Whenreprogramming factors are expressed using non-integrative vectors, thefactors may be expressed in the cells using electroporation,transfection, or transformation of the somatic cells with the vectors.For example, in mouse cells, expression of four factors (Oct3/4, Sox2,c-myc, and Klf4) using integrative viral vectors is sufficient toreprogram a somatic cell. In human cells, expression of four factors(Oct3/4, Sox2, NANOG, and Lin28) using integrative viral vectors issufficient to reprogram a somatic cell.

Once the reprogramming factors are expressed in the cells, the cells maybe cultured. Over time, cells with ES characteristics appear in theculture dish. The cells may be chosen and subcultured based on, forexample, ES morphology, or based on expression of a selectable ordetectable marker. The cells may be cultured to produce a culture ofcells that resemble ES cells—these are putative iPS cells.

To confirm the pluripotency of the iPS cells, the cells may be tested inone or more assays of pluripotency. For examples, the cells may betested for expression of ES cell markers; the cells may be evaluated forability to produce teratomas when transplanted into SCID mice; the cellsmay be evaluated for ability to differentiate to produce cell types ofall three germ layers. Once a pluripotent iPS cell is obtained it may beused to produce RPE cells.

Engineering MHC Genes in Human Embryonic Stem Cells to ObtainReduced-Complexity RPE Cells

Human embryonic stem (hES) cells may be derived from a library of humanembryonic stem cells. The library of human embryonic stern cells maycomprise stem cells, each of which is hemizygous, homozygous, ornullizygous for at least one MHC allele present in a human population,wherein each member of said library of stem cells is hemizygous,homozygous, or nullizygous for a different set of MHC alleles relativeto the remaining members of the library. The library of human embryonicstem cells may comprise stem cells that are hemizygous, homozygous, ornullizygous for all MHC alleles present in a human population. In thecontext of this invention, stem cells that are homozygous for one ormore histocompatibility antigen genes include cells that are nullizygousfor one or more (and in some embodiments, all) such genes. Nullizygousfor a genetic locus means that the gene is null at that locus (i.e.,both alleles of that gene are deleted or inactivated.)

A hES cell may comprise modifications to one of the alleles of sisterchromosomes in the cell's MHC complex. A variety of methods forgenerating gene modifications, such as gene targeting, may be used tomodify the genes in the MHC complex. Further, the modified alleles ofthe MHC complex in the cells may be subsequently engineered to behomozygous so that identical alleles are present on sister chromosomes.Methods such as loss of heterozygosity (LOH) may be utilized to engineercells to have homozygous alleles in the MHC complex. For example, one ormore genes in a set of MHC genes from a parental allele can be targetedto generate hemizygous cells. The other set of MHC genes can be removedby gene targeting or LOH to make a null line. This null line can be usedfurther as the embryonic cell line in which to drop arrays of the HLAgenes, or individual genes, to make a hemizygous or homozygous bank withan otherwise uniform genetic background. Stem cells that are nullizygousfor all MHC genes may be produced by standard methods known in the art,such as, for example, gene targeting and/or loss of heterozygosity(LOH.). See, for example, United States Patent Application Publications2004/0091936, 2003/0217374 and 2003/0232430, and U.S. Provisional PatentApplication No. 60/729,173.

Accordingly, the present invention relates to methods of obtaining RPEcells, including a library of RPE cells, with reduced MHC complexity.RPE cells with reduced MHC complexity may be used to increase the supplyof available cells for therapeutic applications as it may eliminate thedifficulties associated with patient matching. Such cells may be derivedfrom stem cells that are engineered to be hemizygous or homozygous forgenes of the MHC complex.

The invention also provides a library of RPE cells (and/or RPE lineagecells), wherein several lines of ES cells are selected anddifferentiated into RPE cells. These RPE cells and/or RPE lineage cellsmay be used for a patient in need of a cell-based therapy. The inventionalso provides a library of RPE cells, each of which is hemizygous,homozygous, or nullizygous for at least one MHC allele present in ahuman population, wherein each member of said library of RPE cells ishemizygous, homozygous, or nullizygous for a different set of MHCalleles relative to the remaining members of the library. The inventionprovides a library of human RPE cells that are hemizygous, homozygous,or nullizygous for all MHC alleles present in a human population.

Culture Medium

Any medium that is capable of supporting high-density cultures may beused in the methods described herein, such as medium for viral,bacterial, or eukaryotic cell culture. For example, the medium may behigh nutrient, protein-free medium or high nutrient, low protein medium.Further, the medium also may include nutrient components such asalbumin, B-27 supplement, ethanolamine, fetuin, glutamine, insulin,peptone, purified lipoprotein material, sodium selenite, transferrin,vitamin A, vitamin C, or vitamin E. For example, nutrient rich, lowprotein medium may be any medium which supports the growth of cells inculture and has a low protein content. For example, nutrient rich, lowprotein media includes but is not limited to MDBK-GM, OptiPro SFM,VP-SFM, DMEM, RPMI Media 1640, IDMEM, MEM, F-12 nutrient mixture, F-10nutrient mixture EGM-2, DMEM/F-12 media, media 1999, or MDBK-MM. Seealso Table 2. Further, the nutrient rich, low protein medium may be amedium that does not support the growth or maintenance of embryonic stemcells.

When low protein medium is used, the medium may contain at least about20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2.5%, 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25%, 0.20%, 0.10%, 0.05%,0.02%, 0.016%, 0.015%, or 0.010% animal-derived protein (e.g., 10% FBS).Note that reference to the percentage of protein present in low proteinmedium refers to the medium alone and does not account for proteinpresent in, for example, B-27 supplement. Thus, it is understood thatwhen cells are cultured in low protein medium and B-27 supplement, thepercentage of protein present in the medium may be higher.

The low protein or protein free medium are supplemented with serum freeB-27 supplement. Nutrient components of B27 supplement may comprisebiotin, L-carnitine, corticosterone, ethanolamine, D+-galactose, reducedglutathione, linoleic acid, linolenic acid, progesterone, putrescine,retinyl acetate, selenium, triodo-1-thyronine (T3), DL-alpha-tocopherol(vitamin E), DL-alpha-tocopherol acetate, bovine serum albumin,catalase, insulin, superoxide dismutase, and transferrin. When cells arecultured in protein free medium supplemented with B-27, protein freerefers to the medium prior to addition of B-27.

Growth factors, agents, and other supplements described herein may beused alone or in combination with other factors, agents, or supplementsfor inclusion in media. Factors, agents, and supplements may be added tothe media immediately, or any time during or after cell culture.

The medium may also contain supplements such as heparin, hydrocortisone,ascorbic acid, serum (e.g., fetal bovine serum), or a growth matrix(e.g., extracellular matrix from bovine corneal epithelium, MATRIGEL®(basement membrane matrix), or gelatin), fibronectin, proteolyticfragments of fibronectin, laminin, thrombospondin, aggrecan, andsyndezan.

The culture media may be supplemented with one or more factors oragents.

Growth factors that may be used include, for example, EGF, FGF, VEGF,and recombinant insulin-like growth factor. Growth factors that may beused in the present invention also include 6Ckine (recombinant), activinA, α-interferon, alpha-interferon, amphiregulin, angiogenin,β-endothelial cell growth factor, beta cellulin, β-interferon, brainderived neurotrophic factor, cardiotrophin-1, ciliary neurotrophicfactor, cytokine-induced neutrophil chemoattractant-1, endothelial cellgrowth supplement, eotaxin, epidermal growth factor, epithelialneutrophil activating peptide-78, erythropoiten, estrogen receptor-α,estrogen receptor-β, fibroblast growth factor (acidic/basic, heparinstabilized, recombinant), FLT-3/FLK-2 ligand (FLT-3 ligand),gamma-interferon, glial cell line-derived neurotrophic factor,Gly-His-Lys, granulocyte colony-stimulating factor, granulocytemacrophage colony-stimulating factor, GRO-alpha/MGSA, GRO-B, GRO-gamma,HCC-1, heparin-binding epidermal growth factor like growth factor,hepatocyte growth factor, heregulin-alpha (EGF domain), insulin growthfactor binding protein-1, insulin-like growth factor bindingprotein-1/IGF-1 complex, insulin-like growth factor, insulin-like growthfactor II, 2.5 S nerve growth factor (NGF), 7S-NGF, macrophageinflammatory protein-1β, macrophage inflammatory protein-2, macrophageinflammatory protein-3α, macrophage inflammatory protein-3β, monocytechemotactic protein-1, monocyte chemotactic protein-2, monocytechemotactic protein-3, neurotrophin-3, neurotrophin-4, NGF-β (human orrat recombinant), oncostatin M (human or mouse recombinant), pituitaryextract, placenta growth factor, platelet-derived endothelial cellgrowth factor, platelet-derived growth factor, pleiotrophin, rantes,stem cell factor, stromal cell-derived factor 1B/pre-B cell growthstimulating factor, thrombopoetin, transforming growth factor alpha,transforming growth factor-β1, transforming growth factor-β2,transforming growth factor-β3, transforming growth-factor-β5, tumornecrosis factor (α and β, and vascular endothelial growth factor.

Agents that may be used according to the present invention includecytokines such as interferon-α, interferon-α A/D, interferon-β,interferon-γ, interferon-γ-inducible protein-10, interleukin-1,interleukin-2, interleukin-3, interleukin-4, interleukin-5,interleukin-6, interleukin-7, interleukin-8, interleukin-9,interleukin-10, interleukin-11, interleukin-12, interleukin-13,interleukin-15, interleukin-17, keratinocyte growth factor, leptin,leukemia inhibitory factor, macrophage colony-stimulating factor, andmacrophage inflammatory protein-1 α.

The culture media may be supplemented with hormones and hormoneantagonists, including but not limited to 17B-estradiol,adrenocorticotropic hormone, adrenomedullin, alpha-melanocytestimulating hormone, chorionic gonadotropin, corticosteroid-bindingglobulin, corticosterone, dexamethasone, estriol, follicle stimulatinghormone, gastrin 1, glucagon, gonadotropin, hydrocortisone, insulin,insulin-like growth factor binding protein, L-3,3′,5′-triiodothyronine,L-3,3′,5′-triiodothyronine, leptin, leutinizing hormone, L-thyroxine,melatonin, MZ-4, oxytocin, parathyroid hormone, PEC-60, pituitary growthhormone, progesterone, prolactin, secretin, sex hormone bindingglobulin, thyroid stimulating hormone, thyrotropin releasing factor,thyroxine-binding globulin, and vasopressin. The culture media may besupplemented with antibodies to various factors including but notlimited to anti-low density lipoprotein receptor antibody,anti-progesterone receptor, internal antibody, anti-alpha interferonreceptor chain 2 antibody, anti-c-c chemokine receptor 1 antibody,anti-CD 118 antibody, anti-CD 119 antibody, anti-colony stimulatingfactor-1 antibody, anti-CSF-1 receptor/c-fins antibody, anti-epidermalgrowth factor (AB-3) antibody, anti-epidermal growth factor receptorantibody, anti-epidermal growth factor receptor, phospho-specificantibody, anti-epidermal growth factor (AB-1) antibody,anti-erythropoietin receptor antibody, anti-estrogen receptor antibody,anti-estrogen receptor, C-terminal antibody, anti-estrogen receptor-Bantibody, anti-fibroblast growth factor receptor antibody,anti-fibroblast growth factor, basic antibody, anti-gamma-interferonreceptor chain antibody, anti-gamma-interferon human recombinantantibody, anti-GFR alpha-1 C-terminal antibody, anti-GFR alpha-2C-terminal antibody, anti-granulocyte colony-stimulating factor (AB-1)antibody, anti-granulocyte colony-stimulating factor receptor antibody,anti-insulin receptor antibody, anti-insulin-like growth factor-1receptor antibody, anti-interleukin-6 human recombinant antibody,anti-interleukin-1 human recombinant antibody, anti-interleukin-2 humanrecombinant antibody, anti-leptin mouse recombinant antibody, anti-nervegrowth factor receptor antibody, anti-p60, chicken antibody,anti-parathyroid hormone-like protein antibody, anti-platelet-derivedgrowth factor receptor antibody, anti-platelet-derived growth factorreceptor-B antibody, anti-platelet-derived growth factor-alpha antibody,anti-progesterone receptor antibody, anti-retinoic acid receptor-alphaantibody, anti-thyroid hormone nuclear receptor antibody, anti-thyroidhormone nuclear receptor-alpha 1/Bi antibody, anti-transformingreceptor/CD71 antibody, anti-transforming growth factor-alpha antibody,anti-transforming growth factor-B3 antibody, anti-rumor necrosisfactor-alpha antibody, and anti-vascular endothelial growth factorantibody.

Growth medias suitable for use in the methods described herein arelisted in Table 2.

TABLE 2 GROWTH MEDIA FORMULATIONS NAME OF MEDIUM FORMULATION MEF Growth(MEF-GM) 500 mL of IMDM 55 mL FBS hES Growth (hES- GM) 200 mL Knockout ®D-MEM 30 mL Knockout ® Serum Replacement 2 mL GlutaMAX ® -I 2 mL NEAA200 μL 2-mercaptoethanol 10 ng/mL bFGF 10 ng/mL LIF EB Growth (EB-GM) 1L EX-CELL ® MDBK-GM 16.5 mL GlutaMAX ® -I or 1 L OptiPRO-SFM 20 mLG1utaMAX ® -I EB Formation (EB-FM) 1 L EX-CELL ® MDBK-GM 16.5 mLGlutaMAX ® -I 20 mL B-27 Supplement or 1 L OptiPRO-SFM 20 mL GlutaMAX ®-I 20 mL B-27 Supplement RPE Maintenance (RPE-MM) 1 L EX-CELL ® MDBK-MM20 mL GlutaMAX ® -I or 1 L VP-SFM 20 mL GlutaMAX ® -I RPE Growth(RPE-GM) 500 mL EBM ® -2 10 mL FBS 0.2 mL hydrocortisone 2.0 mL rhFGF-B0.5 mL R3-IGF- 1 0.5 mL ascorbic Acid 0.5 mL rhEGF 0.5 mL heparin 0.5 mLVEGFTherapeutic Methods

The RPE cells and pharmaceutically preparations comprising RPE cellsproduced by the methods described herein may be used for cell-basedtreatments. The invention provides methods for treating a conditioninvolving retinal degeneration comprising administering an effectiveamount of a pharmaceutical preparation comprising RPE cells, wherein theRPE cells are derived from pluripotent stem cells in vitro. Conditionsinvolving retinal degeneration include, for example, choroideremia,diabetic retinopathy, retinal atrophy, retinal detachment, retinaldysplasia, and retinitis pigmentosa. The RPE cells described herein mayalso be used in methods for treating macular degeneration including butare not limited to age related macular degeneration (dry or wet), NorthCarolina macular dystrophy, Sorsby's fundus dystrophy, Stargardt'sdisease, pattern dystrophy, Best disease, malattia leventinese, Doyne'shoneycomb choroiditis, dominant drusen, and radial drusen. The RPE cellsdescribed herein may also be used in methods of treating Parkinson'sdisease (PD).

A common feature of cell transplantation is low graft survival, forexample, in many cell transplantation studies there tends to be a lossof cells immediately following transplantation (e.g., within the firstweek). This loss of cells does not appear to be due to rejection of thetransplanted cells but rather an inability of a certain percentage ofthe cells to be retained at the transplant site. This lack of cellretention is most likely due to a number of factors such as the failureof the cells to attach to an underlying structure, a lack of sufficientnutrients, or physical stresses at the transplant site. Following thisinitial drop-off of cell number, the cell survival at various time aftertransplantation can vary considerably from study to study. Thus,although some studies show a steady decline in numbers, other showresults where the grafted cells can reach a stable number. However, animportant factor in considering the success of a transplantation is thepercentage of recipients with surviving grafts following celltransplant.

In contrast with previous preparations, the RPE cells in thepharmaceutical preparations described herein may survive long termfollowing transplantation. For example, the RPE cells may survive atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. Additionally, the RPEcells may survive at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks;at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or months; or at least about1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. Further, the RPE cells maysurvive throughout the lifespan of the receipt of the transplant.Additionally, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97,98, 99, or 100% of the receipts of RPE cells described herein may showsurvival of the transplanted RPE cells. Further, the RPE cells describedherein may successfully incorporate into the RPE layer in thetransplantation receipt, forming a semi-continuous line of cells andretain expression of key RPE molecular markers (e.g., RPE65 andbestrophin). The RPE cells described herein may also attach to theBruch's membrane, forming a stable RPE layer in the transplantationreceipt. Also, the RPE cells described herein are substantially free ofES cells and the transplantation receipts does not show abnormal growthor tumor formation at the transplantation site.

The methods of treating a patient suffering from a condition associatedwith retinal degeneration may comprise administering a composition ofthe invention locally (e.g., by intraocular injection or insertion of amatrix comprising the pharmaceutical preparation of the invention).Intraocular administration of pharmaceutical preparation of theinvention include, for example, delivery into the vitreous body,transcorneally, sub-conjunctival, juxtascleral, posterior scleral, andsub-tenon portions of the eye. See, for example, U.S. Pat. Nos.7,794,704; 7,795,025; 6,943,145; and 6,943,153.

The invention also provides a method of administering human RPE cellsthat have been derived from reduced-complexity embryonic stem cells to apatient. This method may comprise: (a) identifying a patient that needstreatment involving administering human RPE cells to him or her; (b)identifying MHC proteins expressed on the surface of the patient'scells; (c) providing a library of human RPE cells of reduced MHCcomplexity made by the method for producing RPE cells of the presentinvention; (d) selecting the RPE cells from the library that match thispatient's MHC proteins on his or her cells; (e) administering any of thecells from step (d) to said patient. This method may be performed in aregional center, such as, for example, a hospital, a clinic, aphysician's office, and other health care facilities. Further, the RPEcells selected as a match for the patient, if stored in small cellnumbers, may be expanded prior to patient treatment.

The RPE cells may be cultured under conditions to increase theexpression of alpha integrin subunits 1-6 or 9 as compared to unculturedRPE cells or other RPE cell preparations prior to transplantation. TheRPE cells described herein may be cultured to elevate the expressionlevel of alpha integrin subunits 1, 2, 3, 4, 5, 6, or 9. The RPE cellsdescribed herein may be cultured under conditions that promote theexpression of alpha integrin subunits 1-6. For example, the RPE cellsmay be cultured with integrin-activating agents including but notlimited to manganese and the activating monoclonal antibody (mAb)TS2/16. See Afshari, et al. Brain (2010) 133(2): 448-464.

The particular treatment regimen, route of administration, and adjuvanttherapy may be tailored based on the particular condition, the severityof the condition, and the patient's overall health. Administration ofthe pharmaceutical preparations comprising RPE cells may be effective toreduce the severity of the symptoms and/or to prevent furtherdegeneration in the patient's condition. For example, administration ofa pharmaceutical preparation comprising RPE cells may improve thepatient's visual acuity. Additionally, in certain embodiments,administration of the RPE cells may be effective to fully restore anyvision loss or other symptoms. Further, the RPE cell administration maytreat the symptoms of injuries to the endogenous RPE layer.

Pharmaceutical Preparations of RPE Cells

The RPE cells may be formulated with a pharmaceutically acceptablecarrier. For example, RPE cells may be administered alone or as acomponent of a pharmaceutical formulation. The subject compounds may beformulated for administration in any convenient way for use in medicine.Pharmaceutical preparations suitable for administration may comprise theRPE cells, in combination with one or more pharmaceutically acceptablesterile isotonic aqueous or nonaqueous solutions (e.g., balanced saltsolution (BSS)), dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes or suspending or thickening agents.

When administered, the pharmaceutical preparations for use in thisinvention may be in a pyrogen-free, physiologically acceptable form. Thepreparation comprising RPE cells used in the methods described hereinmay be transplanted in a suspension, gel, colloid, slurry, or mixture.Further, the preparation may desirably be encapsulated or injected in aviscous form into the vitreous humor for delivery to the site of retinalor choroidal damage. Also, at the time of injection, cryopreserved RPEcells may be may be resuspended with commercially available balancedsalt solution to achieve the desired osmolality and concentration foradministration by subretinal injection.

The RPE cells of the invention may be delivered in a pharmaceuticallyacceptable ophthalmic formulation by intraocular injection. Whenadministering the formulation by intravitreal injection, for example,the solution may be concentrated so that minimized volumes may bedelivered. Concentrations for injections may be at any amount that iseffective and non-toxic, depending upon the factors described herein.The pharmaceutical preparations of RPE cells for treatment of a patientmay be formulated at doses of at least about 10⁴ cells/mL. The RPE cellpreparations for treatment of a patient are formulated at doses of atleast about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ RPE cells/mL. Forexample, the RPE cells may be formulated in a pharmaceuticallyacceptable carrier or excipient.

The pharmaceutical preparations of RPE cells described herein maycomprise at least about 1,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000;8,000; or 9,000 RPE cells. The pharmaceutical preparations of RPE cellsmay comprise at least about 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴,7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵,8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶,9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10′, 7×10⁷, 8×10⁷, 9×10⁷,1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹,2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰,3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, or 9×10¹⁰ RPE cells. Thepharmaceutical preparations of RPE cells may comprise at least about1×10²-1×10³, 1×10²-1×10⁴-1×10⁵, or 1×10³-1×10⁶RPE cells. Thepharmaceutical preparations of RPE cells may comprise at least about10,000, 20,000, 25,000, 50,000, 75,000, 100,000, 125,000, 150,000,175,000, 180,000, 185,000, 190,000, or 200,000 RPE cells. For example,the pharmaceutical preparation of RPE cells may comprise at least about20,000-200,000 RPE cells in a volume at least about 50-200 μL. Further,the pharmaceutical preparation of RPE cells may comprise at least about180,000 RPE cells in a volume at least about 150 μL.

RPE cells may be formulated for delivery in a pharmaceuticallyacceptable ophthalmic vehicle, such that the preparation is maintainedin contact with the ocular surface for a sufficient time period to allowthe cells to penetrate the affected regions of the eye, as for example,the anterior chamber, posterior chamber, vitreous body, aqueous humor,vitreous humor, cornea, iris/ciliary, lens, choroid, retina, sclera,suprachoridal space, conjunctiva, subconjunctival space, episcleralspace, intracorneal space, epicorneal space, pars plana,surgically-induced avascular regions, or the macula.

The volume of preparation administered according to the methodsdescribed herein may dependent on factors such as the mode ofadministration, number of RPE cells, age and weight of the patient, andtype and severity of the disease being treated. If administered byinjection, the volume of a pharmaceutical preparations of RPE cells ofthe invention may be from at least about 1, 1.5, 2, 2.5, 3, 4, or 5 mL.The volume may be at least about 1-2 mL. For example, if administered byinjection, the volume of a pharmaceutical preparations of RPE cells ofthe invention may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 100, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, or 200 μL (microliters). For example, the volume of a preparationof the invention may be from at least about 10-50, 20-50, 25-50, or1-200 μL. The volume of a preparation of the invention may be at leastabout 10, 20, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, or 200 μL.

For example, the preparation may comprise at least about 1×10³, 2×10³,3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴,4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, or 9×10⁴ RPE cells per μL. Thepreparation may comprise 2000 RPE cells per μL, for example, 100,000 RPEcells per 50 μL or 180,000 RPE cells per 90 μL.

The method of treating retinal degeneration may further compriseadministration of an immunosuppressant. Immunosuppressants that may beused include but are not limited to anti-lymphocyte globulin (ALG)polyclonal antibody, anti-thymocyte globulin (ATG) polyclonal antibody,azathioprine, BASILIXIMAB® (anti-IL-2Rα receptor antibody), cyclosporin(cyclosporin A), DACLIZUMAB® (anti-IL-2Rα receptor antibody),everolimus, mycophenolic acid, RITUXIMAB® (anti-CD20 antibody),sirolimus, and tacrolimus. The immunosuppressants may be dosed at leastabout 1, 2, 4, 5, 6, 7, 8, 9, or 10 mg/kg. When immunosuppressants areused, they may be administered systemically or locally, and they may beadministered prior to, concomitantly with, or following administrationof the RPE cells. Immunosuppressive therapy continues for weeks, months,years, or indefinitely following administration of RPE cells. Forexample, the patient may be administered 5 mg/kg cyclosporin for 6 weeksfollowing administration of the RPE cells.

The method of treatment of retinal degeneration may comprise theadministration of a single dose of RPE cells. Also, the methods oftreatment described herein may comprise a course of therapy where RPEcells are administered multiple times over some period. Exemplarycourses of treatment may comprise weekly, biweekly, monthly, quarterly,biannually, or yearly treatments. Alternatively, treatment may proceedin phases whereby multiple doses are required initially (e.g., dailydoses for the first week), and subsequently fewer and less frequentdoses are needed.

If administered by intraocular injection, the RPE cells may be deliveredone or more times periodically throughout the life of a patient. Forexample, the RPE cells may be delivered once per year, once every 6-12months, once every 3-6 months, once every 1-3 months, or once every 1-4weeks. Alternatively, more frequent administration may be desirable forcertain conditions or disorders. If administered by an implant ordevice, the RPE cells may be administered one time, or one or more timesperiodically throughout the lifetime of the patient, as necessary forthe particular patient and disorder or condition being treated.Similarly contemplated is a therapeutic regimen that changes over time.For example, more frequent treatment may be needed at the outset (e.g.,daily or weekly treatment). Over time, as the patient's conditionimproves, less frequent treatment or even no further treatment may beneeded.

The methods described herein may further comprises the step ofmonitoring the efficacy of treatment or prevention by measuringelectroretinogram responses, optomotor acuity threshold, or luminancethreshold in the subject. The method may also comprise monitoring theefficacy of treatment or prevention by monitoring immunogenicity of thecells or migration of the cells in the eye.

The RPE cells may be used in the manufacture of a medicament to treatretinal degeneration. The invention also encompasses the use of thepreparation comprising RPE cells in the treatment of blindness. Forexample, the preparations comprising human RPE cells may used to treatretinal degeneration associated with a number of vision-alteringailments that result in photoreceptor damage and blindness, such as,diabetic retinopathy, macular degeneration (including age-relatedmacular degeneration, e.g., wet age-related macular degeneration and dryage-related macular degeneration), retinitis pigmentosa, and Stargardt'sDisease (fundus flavimaculatus). The preparation may comprise at leastabout 5,000-500,000 RPE cells (e.g., 100,00 RPE cells) which may beadministered to the retina to treat retinal degeneration associated witha number of vision-altering ailments that result in photoreceptor damageand blindness, such as, diabetic retinopathy, macular degeneration(including age-related macular degeneration), retinitis pigmentosa, andStargardt's Disease (fundus flavimaculatus).

The RPE cells provided herein may be human RPE cells. Note, however,that the human cells may be used in human patients, as well as in animalmodels or animal patients. For example, the human cells may be tested inmouse, rat, cat, dog, or non-human primate models of retinaldegeneration. Additionally, the human cells may be used therapeuticallyto treat animals in need thereof, such as in veterinary medicine.

Modes of Administration

The pharmaceutical preparation may be formulated in a pharmaceuticallyacceptable carrier according to the route of administration. Forexample, the preparation may be formulated to be administered to thesubretinal space of the eye. The preparation comprising RPE cells may beadministered to one eye or both eyes in the same patient. Theadministration to both eyes may be sequential or simultaneous. Forexample, the preparation comprising RPE cells may be formulated as asuspension, solution, slurry. gel, or colloid.

RPE cells of the invention may be administered locally by injection(e.g., intravitreal injection), or as part of a device or implant (e.g.,an implant). For example, the preparation may be administered byinjection into the subretinal space of the eye. Also, the preparationmay be administered transcorneally. For example, the cells of thepresent invention may be transplanted into the subretinal space by usingvitrectomy surgery. Additionally, at the time of injection, RPE cellsmay be may be resuspended with commercially available balanced saltsolution to achieve the desired osmolality and concentration foradministration by subretinal injection.

Depending on the method of administration, the RPE cells may be added tobuffered and electrolyte balanced aqueous solutions, buffered andelectrolyte balanced aqueous solutions with a lubricating polymer,mineral oil or petrolatum-based ointment, other oils, liposomes,cylcodextrins, sustained release polymers or gels.

Matrices for Use with RPE Cells

The methods described herein may comprise a step of administering RPEcells of the invention as an implant or device. In certain embodiments,the device is bioerodible implant for treating a medical condition ofthe eye comprising an active agent dispersed within a biodegradablepolymer matrix, wherein at least about 75% of the particles of theactive agent have a diameter of less than about 10 μm. The bioerodibleimplant is sized for implantation in an ocular region. The ocular regionmay be any one or more of the anterior chamber, the posterior chamber,the vitreous cavity, the choroid, the suprachoroidal space, theconjunctiva, the subconjunctival space, the episcleral space, theintracorneal space, the epicorneal space, the sclera, the pars plana,surgically-induced avascular regions, the macula, and the retina. Thebiodegradable polymer may be, for example, apoly(lactic-co-glycolic)acid (PLGA) copolymer, biodegradablepoly(DL-lactic-co-glycolic acid) films, or PLLA/PLGA polymer substrates.The ratio of lactic to glycolic acid monomers in the polymer is about25/75, 40/60, 50/50, 60/40, 75/25 weight percentage, more preferablyabout 50/50. The PLGA copolymer may be about 20, 30, 40, 50, 60, 70, 80to about 90 percent by weight of the bioerodible implant. The PLGAcopolymer may be from about 30 to about 50 percent by weight, preferablyabout 40 percent by weight of the bioerodible implant. The RPE cells maybe transplanted in conjunction with a biocompatible polymer such aspolylactic acid, poly(lactic-co-glycolic acid), 50:50 PDLGA, 85:15PDLGA, and INION GTR® biodegradable membrane (mixture of biocompatiblepolymers). See U.S. Pat. Nos. 6,331,313; 7,462,471; and 7,625,582. Seealso Hutala, et al. (2007) “In vitro biocompatibility of degradablebiopolymers in cell line cultures from various ocular tissues: Directcontact studies.” Journal of Biomedical Materials Research 83A(2):407-413; Lu, et al. (1998) J Biomater Sci Polym Ed 9: 1187-205; andTomita, et al. (2005) Stem Cells 23: 1579-88.

Screening Assays

The invention provides a method for screening to identify agents thatmodulate RPE cell maturity. For example, RPE cells differentiated fromhuman ES cells may be used to screen for agents that promote RPEmaturation. Identified agents may be used, alone or in combination withRPE cells, as part of a treatment regimen. Alternatively, identifiedagents may be used as part of a culture method to improve the survivalof RPE cells differentiated in vitro.

The RPE cells may be used a research tool in settings such as apharmaceutical, chemical, or biotechnology company, a hospital, or anacademic or research institution. Such uses include the use of RPE cellsdifferentiated from embryonic stem cells in screening assays toidentify, for example, agents that may be used to promote RPE survivalin vitro or in vivo, or that may be used to promote RPE maturation.Identified agents may be studied in vitro or in animal models toevaluate, for example, their potential use alone or in combination withRPE cells.

The invention provides a method for identifying agents that promote RPEmaturation comprising providing a RPE cell, contacting said RPE cellwith an agent, assessing said RPE cell for signs of maturity, and thenidentifying an agent that promotes RPE maturation when said agent causesRPE cell to show signs of maturity. The signs of maturity may bepigmentation level, gene expression levels, and morphology as discussedherein.

Commercial Applications and Methods

Certain aspects of the present invention pertain to the production ofRPE cells to reach commercial quantities. The RPE cells may be producedon a large scale, stored if necessary, and supplied to hospitals,clinicians or other healthcare facilities.

Accordingly certain aspects of the present invention relate to methodsof production, storage, and distribution of RPE cells produced by themethods disclosed herein. Following RPE production, RPE cells may beharvested, purified, and optionally stored prior to a patient'streatment. RPE cells may optionally be patient specific or specificallyselected based on HLA or other immunologic profile. For example, once apatient presents with an indication such as, for example, diabeticretinopathy, macular degeneration (including age-related maculardegeneration), retinitis pigmentosa, retinal atrophy, retinaldetachment, retinal dysplasia, and Stargardt's Disease (fundusflavimaculatus), RPE cells may be ordered and provided in a timelymanner. Accordingly, the present invention relates to methods ofproducing RPE cells to attain cells on a commercial scale, cellpreparations comprising RPE cells derived from said methods, as well asmethods of providing (i.e., producing, optionally storing, and selling)RPE cells to hospitals and clinicians. The production of differentiatedRPE cells or mature differentiated RPE cells may be scaled up forcommercial use.

The present invention also provides for methods of conducting apharmaceutical business comprising establishing a distribution systemfor distributing the preparation for sale or may include establishing asales group for marketing the pharmaceutical preparation.

The present invention provides methods of supplying RPE cells tohospitals, healthcare centers, and clinicians, whereby RPE cellsproduced by the methods disclosed herein are stored, ordered on demandby a hospital, healthcare center, or clinician, and administered to apatient in need of RPE cell therapy. A hospital, healthcare center, orclinician orders RPE cells based on patient specific data, RPE cells areproduced according to the patient's specifications and subsequentlysupplied to the hospital or clinician placing the order. For example,after a particular RPE cell preparation is chosen to be suitable for apatient, it is thereafter expanded to reach appropriate quantities forpatient treatment.

Further aspects of the invention relate to a library of RPE cells thatcan provide matched cells to potential patient recipients. Accordingly,the invention provides a method of conducting a pharmaceutical business,comprising the step of providing RPE cell preparations that arehomozygous for at least one histocompatibility antigen, wherein cellsare chosen from a bank of such cells comprising a library of RPE cellsthat may be expanded by the methods disclosed herein, wherein each RPEcell preparation is hemizygous or homozygous for at least one MHC allelepresent in the human population, and wherein said bank of RPE cellscomprises cells that are each hemizygous or homozygous for a differentset of MHC alleles relative to the other members in the bank of cells.As mentioned above, gene targeting or loss of heterozygosity may be usedto generate the hemizygous or homozygous MHC allele stem cells used toderive the RPE cells.

The present invention also includes methods of obtaining human ES cellsfrom a patient and then generating and expanding RPE cells derived fromthe ES cells. These RPE cells may be stored. In addition, these RPEcells may be used to treat the patient from which the ES were obtainedor a relative of that patient.

The present disclosure demonstrates that human RPE cells may be reliablydifferentiated and expanded from human ES cells under well-defined andreproducible conditions—representing an inexhaustible source of cellsfor patients with retinal degenerative disorders. The concentration ofthese cells would not be limited by availability, but rather could betitrated to the precise clinical requirements of the individual.Repeated infusion or transplantation of the same cell population overthe lifetime of the patient would also be possible if deemed necessaryby the physician. Furthermore, the ability to create banks of matchingor reduced-complexity HLA hES lines from which RPE cells could beproduced could potentially reduce or eliminate the need forimmunosuppressive drugs and/or immunomodulatory protocols altogether.

The present invention will now be more fully described with reference tothe following examples, which are illustrative only and should not beconsidered as limiting the invention described above.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Method of Making Human RPE Cells Using HES Cells

Mouse embryo fibroblasts (MEF) were grown in MEF-GM medium supplementedwith about 10% fetal bovine serum (FBS). When sufficient numbers of MEFswere obtained, feeder cells were prepared by mitotically blocking theMEFs with mitomycin-C and seeding into 6-well plates coated withgelatin. See FIG. 1. Vials of hES were thawed, seeded on to the MEFfeeder cells, and co-cultured in hES Growth Medium. See Table 2 and FIG.2. The hES cells were expanded several times at a split ratio of about1:3. When a sufficient number of hES cells were propagated, the cellswere harvested and placed into suspension culture in low attachment6-well plates in EB Formation Medium (this allows for the formation ofembryoid bodies (EBs)). See Table 2 and FIG. 3.

The EBs were then be transferred to gelatin-coated 6-well plates toallow for the outgrowth of RPEs. The initial growth medium is EBOutgrowth Medium, but once the EBs were attached this was changed to EBMaintenance Medium. See Table 2. When cultures were about 70% confluentthe medium was changed to MDBK-MM. Once sufficient numbers of RPE cellclusters were visible, the RPE cells were isolated and furtherpropagated in EGM-2 medium until confluent. When confluent the RPE cellswere cultured in MDBK-MM until the cells reach a medium pigmentmorphology and pigmentation. See FIGS. 4 and 5. The RPE cells were thenharvested and stored frozen at below about −135° C. (e.g., in the vaporphase of liquid nitrogen). See FIG. 6. The RPE cells were produced incompliance with GMP. Thus, this method yields an effective amount ofhuman RPE cells suitable for use in transplantation.

Example 2 Seeding and Expansion of hES Cells

Cryopreserved human embryonic stem cells (hES) cells were thawed, washedwith hES-GM, and inoculated onto the mitotically inactivated mouseembryonic feeder (MEF) cells in the gelatin-coated 6-well plates. SeeFIG. 7. The contents of each vial (˜1 million cryopreserved cells) ofhES were seeded into one well of a 6-well plate, and co-cultures of hESand MEF are incubated for about 4-9 days until about 60-80% confluent.During this time the cultures were examined microscopically: largercolonies displaying mostly hES morphology were dispersed into smallerpieces to prevent spontaneous differentiation. Mosaic colonies withlarge areas of undifferentiated cells were trimmed by removing thoseportions comprised of differentiated cells. Colonies containingpredominately differentiated cells or non hES cell morphology werepicked and discarded, using a stem cell cutting tool using photographsas a guide to the morphology of the colonies. See FIG. 2.

When 60-80% confluent, the hES cells were passaged by washing withCa²⁺/Mg²⁺-free DPBS and treated with 0.05% trypsin/EDTA for about 2-5minutes until detached. The trypsin was neutralized with MEF-GM and thecells collected by centrifugation. The hES cells are then reseeded onfresh MEF feeder layers. The hES cells were expanded several times at asplit ratio of about 1:4 or less. See FIG. 3.

When a sufficient number of hES cells were propagated, hES cells wereharvested. The cells were wash with Ca²⁺/Mg²⁺-free DPBS and treated with0.05% trypsin/EDTA for about 2-5 minutes until detached. The trypsin wasneutralized with MEF-GM and the cells collected by centrifugation. ThehES cells were then resuspended in EB Formation Medium (EB-FM).

No additional MEF cells were used. The time between MEF removal and RPEcells harvest was 3 passages and about 80-90 days in MEF-free cellculture. The hES cell made by this method were further tested andconfirmed to be substantially free of MEF cells by, for example,assaying for mouse specific markers. Upon testing of hES cell made bythis method, it was found that the hES cells were substantially free ofMEF cells.

Example 3 Human Embryoid Body Formation and Outgrowth

hES cells were inoculated onto low-attachment, 6-well plates (at a splitratio of 1:2) and cultured for about 7-12 days until embryoid bodieswere formed and matured. Embryoid bodies in suspension were harvestedfrom the low attachment wells, resuspended in EB-FM, and plated ontogelatin-coated 6-well culture plates. The plates were culturedundisturbed for about 3-4 days to allow the embryoid bodies to attach.At this time, medium was changed to EB Growth Medium (EB-GM). Whencultures were about 70% confluent (e.g., after about 9-12 days), themedium was changed to RPE Maintenance Medium (RPE-MM). See FIG. 3. Celloutgrowths from the attached EBs were sustained in culture in RPE-MMuntil the appropriate number of pigmented clusters were visible (e.g.,after about 35-50 days after changing to RPE-MM). Thus, embryoid bodieswere formed and isolated substantially free of non-human cells and thusnot “xenotransplantation” material. These EB may then be differentiatedto produce human RPE cells.

Example 4 Use of Cryopreserved hES Cells for Human Embryoid Bodies

Cryopreserved hES cells (e.g., MA01 and MA09) were thawed and placedinto suspension culture on Lo-bind Nunclon Petri dishes in MDBK-GrowthMedium or OptimPro SFM supplemented with L-Glutamine,Penicillin/Streptomycin, and B-27 supplement. The hES cells had beenpreviously derived from single blastomeres biopsied from early cleavagestage human embryos. The remainder of the human embryo was notdestroyed. The cells were cultured for at least about 7-14 days asembryoid bodies (EBs).

After at least about 7-14 days, the EBs were plated onto tissue cultureplates coated with gelatin from porcine skin. The EBs were grown asadherent cultures for an at least about an additional 14-28 days inMDBK-Growth Medium or OptimPro SFM supplemented with L-Glutamine, andPenicillin/Streptomycin, without B-27 supplement. From amongst the cellsin the adherent culture of EBs, RPE cells became visible and wererecognized by their cobblestone cellular morphology and emergence ofpigmentation. Therefore, cryopreserved hES cells may be thawed,cultured, and used to form EBs that may, in turn, be used to producehuman RPE cells without the use of MEF cells.

Example 5 Human RPE Cell Derivation

Human RPE cell derivation was initiated when about 10-30 patches oflight to dark brown clusters ˜1 mm or less in diameter were visible ineach well. This may require about 40-50 days after switching to RPE-MM.Embryoid body cellular outgrowths containing pigmented patches wereharvested by incubating in Type IV collagenase in DPBS with Ca²⁺/Mg²⁺until cell clusters have detached. The detached cell clusters weretriple washed in RPE-MM and transferred to 100 mm non-tissue (ultra lowattachment) culture dishes. Under a stereomicroscope, clusters ofpigmented cells were mechanically separated from non-pigmented cellclusters using a stem cell cutting tool. Once all the pigmented clustershave been isolated, the collected clusters were examined under thestereomicroscope to remove any non-pigmented clusters that may have beentransferred. Pigmented cell clusters were washed in RPE-MM and thendissociated in a 1:1 mix of 0.25% Trypsin-EDTA and Cell DissociationBuffer. The dissociated cells were washed in MEF-GM to neutralize thetrypsin and centrifuged. Cell pellets were resuspended in RPE GrowthMedium (RPE-GM) before plating in gelatin-coated 6-well plates. See FIG.4. Accordingly, cultures of human RPE cells that are substantially freeof hES cells may be differentiated and isolated without the use ofnon-human feeder cells. Therefore, the human RPE cells prepared inaccordance with the methods described herein may be consideredsubstantially free of non-human cells, thus not a xenotransplantationmaterial, and hES cells, thus not tumorigenic.

Example 6 Human RPE Expansion

Resuspended human RPE cells were inoculated onto gelatin-coated 4-wellor 6-well plates at a density of 50,000 or 250,000 cells, respectivelyin RPE-GM and cultured until confluent (about 8-11 days). At this time,the medium was changed to RPE-MM and incubated for about 9-14 days untilthe RPE cultures display a medium level of pigmentation. Passage 0 (P0)RPE cultures were harvested with a 1:1 mix of 0.25% Trypsin-EDTA andCell Dissociation Buffer, neutralized with MEF-GM and collected bycentrifugation. Cell pellets were resuspended in RPE-GM and reinoculatedonto gelatin-coated plates at a ratio of 1:3 to 1:6. RPE cell cultureswere expanded at least twice (undergoing two 1:3 to 1:6 splits (passage2 designation). At this time the human RPE cells were harvested. In thismanner, the number of RPE cells may be greatly increased including toreach therapeutically useful amounts of human RPE cells (e.g., at leastabout 1×10³-10⁶RPE cells).

Example 7 Propagation of Mature Human RPE Cells

RPE cells were cultured in an adherent culture. As differentiated RPEcells appear in the adherent cultures, clusters of differentiated RPEsmay become visibly noticeable based on cell shape. Frozen collagenase IV(20 mg/ml) was thawed and diluted to 7 mg/ml. The collagenase IV wasapplied to the adherent culture containing RPE clusters (1.0 ml to eachwell in a 6-well plate). Over about 1-3 hours, the collagenase IVdissociated the cell clusters. By dissociating the RPE clusters fromother cells in the culture, an enriched suspension of RPE cells wasobtained. The enriched RPE cell suspension was removed from the cultureplate and transferred to a 100 mm tissue culture dish with 10 ml of MEFmedium. Pigmented clumps were transferred with a stem cell cutting tool(Swemed-Vitrolife) to a well of a 6-well plate containing 3 ml of MEFmedia. After all clumps have been picked up, the suspension of pigmentedcells was transferred to a 15 ml conical tube containing 7 ml of MEFmedium and centrifuged at 1000 rpm for five minutes. The supernatant wasremoved. 5 ml of a 1:1 mixture of 0.25% trypsin and cell dissociationbuffer was added to the cells. The cells were incubated for 10 minutesat 37° C. The cells were dispersed by pipetting in a 5 ml pipette untilfew clumps were remaining. 5 ml of MEF medium was added to the cells andthe cells centrifuged at 1000 rpm for 5 minutes. The supernatant wasremoved and the cells were plated on gelatin coated plates with a splitof 1:3 of the original culture in EGM-2 culture medium. See FIG. 4.

The culture of RPE cells was expanded by continued culture in EGM-2medium. The cells were passaged, as necessary, at a 1:3 to 1:6 ratiousing a 1:1 mixture of 0.25% trypsin EDTA and Cell Dissociation Buffer.To enrich for mature differentiated RPE cells, the cells were grown tonear confluency in EGM-2. The medium was then changed to MDBK-MM (SAFCBiosciences) to further promote maturation of the RPE cells.Accordingly, mature human RPE cells may be prepared for use intherapeutic methods.

Example 8 RPE Cells Harvest and Cryopreservation

The human RPE cells were grown to near confluency and the medium changedto RPE Maintenance Medium. The RPE cells were then cultured until thecells reach a medium pigment morphology and pigmentation. This may takeat least about one additional month of culture time. The medium pigmentis based on a culture that appears to contain about half of the cells inthe dense cobblestone state and half the culture in the lighter, lessdense morphology. Pictures may be utilized to help standardize theprocess. The medium pigment morphology was chosen because the viabilitypost-thaw is maintained, the recovery of the cells is better than thehigh pigment, and the pharmacology showed similar efficacy to the othermorphologies.

P2 or P3 medium-pigmented RPE cells in culture were harvested by washingand treatment with 0.25% Trypsin-EDTA. Detached RPE cells were washedwith MEF-GM to neutralize the trypsin, centrifuged, counted andresuspended in a solution of 90% FBS and 10% DMSO at a concentration of1 million cells/mL. One mL of cell product suspension was dispensed intoan appropriately labeled, sterile, 1.2 mL cryovials. Vials were storedfor 1-3 days at −80° C. prior to transfer to the vapor phase of liquidnitrogen storage (−135° C.) See FIG. 6. Thus the cryopreservedpreparations of RPE cells may be manufactured.

Example 9 Compliance with GTP and/or GMP Regulations

Human RPE cells, either harvested or thawed from cryopreserved vials maybe tested and characterized in compliance with GTP and/or GMPRegulations as presented in Table 3. See also 21 C.F.R. § 210 and § 211.

TABLE 3 Release Specifications for RPE cells Test Method SpecificationSterility USP Negative Mycoplasma Direct culture Negative Hoechst stainNegative Mouse DNA PCR Negative Mouse Antibody Inoculation into micewith LCMV Negative Production challenge for antibodies to 19 virusesplus LDHE and LC viruses Endotoxin Endotoxin specific turbidimetricmethod <0.50 EU/ml In vitro viruses Indicator Cells—cytopathic effectNegative Indicator Cells—hemadsorption Negative IndicatorCells—hemagglutination Negative Inoculation into suckling mice NegativeInoculation into adult mice Negative Inoculation into embryonated henNegative eggs allantoic route Inoculation into embryonated hen Negativeeggs—yolk sac route Viability Trypan blue dye exclusion >85% Karyotype Gbanding with FISH Normal 46 XX Morphology Visual examination Confluent,RPE at harvest morphology, medium pigmentation Presence of qPCR Allpresent RPE Markers Bestrophin RPE-65 CRALBP PEDF PAX6 MITF Absence ofqPCR All absent hES markers Oct-4 NANOG Rex-1 Sox2 Presence ofImmunostaining All present RPE Markers Bestrophin CRALBP PAX6 MITF ZO-1Absence of Immunostaining All absent hES Markers Oct-4 Alkalinephosphatase Potency Phagocytosis assay Positive Purity Immunostainingfor PAX6 >95% staining and MITF for PAX6 and/or MITF Immunostaining forPAX6 >95% staining and bestrophin for PAX6 and/or bestrophinImmunostaining for ZO-1 >95% staining Viability Trypan blue exclusion>85% Prior to >70% cryopreservation Post cryopreservation

Table 4 provides a description of the tests that may be performed forcharacterization and qualification during the production of RPE cellsincluding RPE cells preparations for use in transplantation therapies.The RPE cells produced in accordance with the methods described hereinmay be tested by at least one of the tests listed in Table 4.

TABLE 4 Description of Tests for Characterization and QualificationManufacturing Step Test Where Performed Assay and Description SterilitySterility Release of MEF cells This assay may detect the presence of oneor more species of Release of hES seed bank bacterial and fungalcontaminants in the test article. This Release of hES MCB determinationis made using a membrane filtration meeting (Master Cell Bank) USP <71>.Bacteriostasis and fungistasis testing may also included. MycoplasmaMycoplasma Release of MEF cells This assay may be used to determine thepresence of Mycoplasma in Release of hES seed bank the test articlebased on the ability of Mycoplasma to grow in one of Release of hES MCBthe test systems. In the direct culture procedure, broth and two typesRelease of Product of agar plates are inoculated and incubatedaerobically and microaerophilically. The inoculated broth bottles aresubcultured on three separate occasions onto agar plates. All plates areexamined for the presence of Mycoplasma colonies at least 14 days afterinoculation. In addition the test article is inoculated into VERO cellcultures, incubated for three to five days and then stained usingHoechst DNA fluorochrome stain. These stained cultures are then examinedmicroscopically. Purity (Endotoxin) Endotoxin Release of Product Thisassay may detect and quantify gram negative bacterial endotoxins(lipopolysaccharides) using an endotoxin specific turbidimetric method.After a minimum of 2 days in liquid nitrogen, product vials are thawedand formulated as per BR @ 1333 viable RPE/μL. Purity (OtherContaminants) Retrovirus detection using feline Release of MEF cellsThis assay may detect replication competent retroviruses. In the PG-4(S*L) cells Release of hES MCB direct assay the MEF cells are inoculateddirectly feline PG-4 (S*L) cells prepared in the presence of 4 μg/mL ofpolybrene and allowed to absorb for 60-90 minutes. After the absorptionperiod, growth medium was added to the cells. And foci were enumeratedon Day 5. In the amplified assay the test cells were inoculated intoflasks of Mus Dunni cells treated with 8 μg/mL of polybrene. The cellsare cultured for 14 days, with subpassage as needed. Culture fluids wereharvested after the final sub pass and inoculated onto feline PG-4 (S*L)cells as described for the direct assay. Viral reverse transcriptaseRelease of MEF cells This assay may detect the presence of retrovirusesby detecting viral detection Release of hES MCB reverse transcriptaseactivity using a PCR-based assay. If reverse transcriptase is present,RNA is reverse transcribed to cDNA. After reverse transcription, thesamples are subjected to PCR and amplification monitored by fluorescencedetection. The assay may detect as few as 55 viral particles.Ultrastructural evaluation for Release of MEF cells This assay may beused to examine 200 test article cells using virus-like particlesRelease of hES MCB transmission electron microscopy techniques todetermine if virus- like particles are present. If retrovirus-likeparticles are present, the cells are evaluated for particle morphology(A, B, C, D, or R-type) and the number of cells with retrovirus-likeparticles is tabulated. In vitro viral agents Release of MEF This assaymay be used to determine whether adventitious viral Release of hES MCBcontaminants are present in cells by direct inoculation and observationof indicator cells for cytopathic effects, hemadsorption, andhemagglutination. The indicator cell lines used were MRC-5 (humandiploid lung cells), Vero 76 (African green monkey kidney cells), andHeLa (human epithelioid carcinoma cells). This assay includedsub-cultures for an additional 14 days. Hemagglutination andhemadsorption was tested using chicken, guinea pig and human Oerythrocytes. Detection of inapparent viruses Release of MEF This assaymay detect viruses present in the test article, which do not Release ofhES MCB have a discernable effect in cell culture systems. The testarticle is inoculated into adult and suckling mice, guinea pigs, andembryonated hen's eggs (yolk sac and allantoic route). Suckling mice aresubpassaged. All animals are observed for signs of illness and any thatbecome sick are examined in an attempt to establish the cause of illnessor death. Allantoic fluid and yolk sac are subpassaged. Both direct andsubpassaged allantoic fluids are tested for hemagglutination temperatureusing guinea pig, human type O, and chick erythrocytes. Embryos may beexamined for viability. Detection of MVM Release of MEF This assay maydetect the presence of MVM DNA sequences in the MEF cells. Genomic DNAwas isolated from MEF cells. The DNA was amplified using PCR withprimers and fluorescent probes specific for MVM and with fluorescencedetection of amplification. Murine ecotropic viruses Release of hES MCBThis assay may detect the presence of infectious ecotropic murineleukemia retroviruses (E-MuLv). The assay utilizes a mixed culture ofSC-1 cells (feral mouse) and XC cells (derived from a Wistar rat tumorinduced by Rous Sarcoma virus). The assay is designed such that SC-1cells (pretreated with polybrene). E-MuLv is capable of replicating inthe SC-1 cells, however no morphological changes are observed. Afterseveral passages XC cells are placed in contact with the infected SC-1cells. The XC undergo characteristic morphological changes (syncytiumformation) due to E-MuLv infection. Bovine viruses Release of hES MCBThis assay may detect bovine viruses that may be cultured with BT(bovine turbinate) and Vero cells. The detection of these viruses isbased upon microscopic observation of viral cytopathic effects in theindicator cells, immunofluorescent staining with virus-specificantibodies for viruses (bovine viral diarrhea virus, bovine parvovirus,bovine adenovirus, bovine respiratory syncytial virus, reovirus, bluetongue virus, and rabies virus), and a hemadsorption assay using chickenand guinea pig erythrocytes. Porcine Viruses Release of hES MCB Thisassay may detect porcine viruses that may be by cultured with ST (swinetestis) and Vero cells. The detection of these viruses is based uponmicroscopic observation of viral cytopathic effects in the indicatorcells, immunofluorescent staining with virus-specific antibodies forviruses (bovine viral diarrhea virus, porcine parvovirus, porcineadenovirus, reovirus, transmissible gastroenteritis virus (also known asporcine coronavirus type 1), hemaggluting encephalitis virus, and rabiesvirus), and a hemadsorption assay using chicken and guinea pigerythrocytes. HIV-1 virus Release of hES MCB This assay may detect thepresence of HIV-1 sequences in the test article (genomic DNA, 0.5 μg) byPCR amplification using HIV-1- specific primers. The amplificationproducts are then analyzed by fluorescence. HIV-2 virus Release of hESMCB This assay may detect the presence of HIV-2 sequences in the testarticle (genomic DNA, 0.5 μg) by PCR amplification using HIV-2 -specific primers. The amplification products are then analyzed byfluorescence. Human Cytomegalovirus Release of hES MCB This assay maydetect the presence of CMV sequences in the test article (genomic DNA,0.5 μg) by PCR amplification using CMV- specific primers. Theamplification products are then analyzed The amplification products arethen analyzed by fluorescence. Human Parvovirus B19 Release of hES MCBThis assay may detect the presence of HumanParvovirus B19 sequences inthe test article (genomic DNA, 0.5 μg) by PCR amplification using HumanParvovirus B19-specific primers. The amplification products are thenanalyzed by fluorescence. Human Epstein-Barr virus Release of hES MCBThis assay may detect the presence of EBV sequences in the test article(genomic DNA, 0.5 μg) by PCR amplification using EBV- specific primers.The amplification products are then analyzed by fluorescence. HumanHepatitis B virus Release of hES MCB This assay may detect the presenceof HBV sequences in the test article (genomic DNA, 0.5 μg) by PCRamplification using HBV- specific primers. The amplification productsmay then be analyzed by fluorescence. Human Hepatitis C Virus Release ofhES MCB This assay may detect the presence of HCV sequences in the testarticle (genomic DNA, 0.5 μg) by PCR amplification using HCV- specificprimers. The amplification products may then be analyzed byfluorescence. Human Herpesvirus 6 Variant A Release of hES MCB Thisassay may detect the presence of HHV-6A and HHV-6B (HHV-6A) and B(HHV-6B) sequences in the test article (genomic DNA, 0.5 μg) by PCRamplification using HHV-6A and HHV-6B-specific primers. Theamplification products may then be analyzed by fluorescence. HumanT-cell Lymphotropic Release of hES MCB This assay may detect thepresence of HTLV-1 and HTLV-2 Virus 1 and 2 (HTLV-1 and -2) sequences inthe test article (genomic DNA, 0.5 μg) by PCR amplification using HTLV-1and HTLV-2-specific primers. The amplification products may then beanalyzed by fluorescence. Mouse antibody production Release of hES MCBThis assay may detect the presence of murinc viruses. The hES cells wereadministered to mice (intraperitoneally, intranasaly, and per os) onDay 1. Nine days later blood samples were collected from a subset formeasurement of lactate dehydrogenase activity. On Day 23, a furthersubset of mice was challenged with a lethal dose of lymphocyticchoriomeningitis virus (LCMV). Finally on Day 28 serum was collectedfrom the remaining animals for assay of viral antibodies. Antibodieswere detected by ELISA of IFA. Cell Purity Release of Product This assaymay detect the presence of RPE markers using immunostaining for MITF,PAX6 and DAPI. After cryopreservation, vials of Product may be thawedand formulated in the clinical diluent. The cells may then becentrifuged, resuspended in RPE-GM and plated onto gelatin-coated platesand cultured. The cells may be stained 1-3 days post-seeding. IdentityGene Expression Release of hES MCB This assay may be used to determinethe gene expression in the cells and compare to the morphologicalfeatures Human Affymetrix HG-U133 Plus 2.0 microarray platform andperformed subsequent informatic analysis Karyotype Release of hES MCBThis assay may be used to confirm that the cells have maintained a Inprocess testing normal chromosomal complement. It may be done usingG-hand (hES Cell expansion) staining with FISH Release of ProductMorphology In process. This assay may be used to confirm that the cellsare of morphology Release of Product consistent with RPE cells. Presenceof ES markers (q-PCR) Release of hES MCB This assay may detect thepresence of ES markers Rex1, NANOG, TDGF-1, Sox-2, DPPA-2, DPPA-4 byRT-qPCR for RPE mRNA expression. The amplification products may then beanalyzed by fluorescence. Presence of RPE markers Release of ProductThis assay may detect the presence of RPE markers bestrophin, (q-PCR)RPE-65, CRALBP, PEDF, PAX6, MITF, ZO-1) by RT-qPCR for RPE mRNAexpression. The amplification products may then be analyzed byfluorescence. After a minimum of 2 days in LN2, the cryopreservedproduct vials are thawed and formulated in the clinical diluent. RNAfrom the cells is extracted extract RNA used to generate cDNA andanalyzed. Presence of RPE markers Release of Product This assay maydetect lhe presence of RPE markers bestrophin, (immunostaining) RPE-65,CRALBP, PEDF, PAX6, MITF, ZO-1) by immunostaining After a minimum of 2days in LN2, the cryopreserved product vials are thawed and formulatedin the clinical diluent. The cells may then be centrifuged, resuspendedin RPE-GM and plated onto gelatin- coated plates and cultured. The cellsmay be stained 2-3 weeks after plating to mature into medium-pigmentedRPE. Absence of ES markers (q-PCR) Release of Product This assay maydetect the absence of ES specific markers by PCR amplification. Theamplification products may then be analyzed by fluorescence. After aminimum of 2 days in LN2, the cryopreserved product vials are thawed andformulated in the clinical diluent. RNA from the cells is extractedextract RNA used to generate cDNA and analyzed. Absence of ES markersRelease of Product This assay may be used to confirm that no ES cellsare absent in the (immunostaining) Cell Product markers. Cells arecollected prior to last centrifugation and addition of cryopreservationmedium. The cells may be seeded into gelatin-coated and cultured until30-60% confluent. The cells may then be stained. Potency Potency Releaseof Product This assay may be used to characterize the RPE product usinga RPE- specific phagocytosis. Cells may be seeded in gelatin-coaledwells of a 96-well plate and grown in RPE-MM until medium-pigmented. Thecells may then be incubated fluorescent polystyrene. The plates wereincubated for 24-36 hours at 37° C. Negative control was performed withthe same plates incubated at 1-4° C. for the same duration. After theincubation, the plates were rinsed 3 times with PBS to remove theremaining particles. fixed with 2% paraformaldehyde in PBS, rinsed twicewith PBS and examined and photographed under the fluorescence invertedmicroscope. Cell Viability Viability Harvest and cryo- This assay may beused to measure the number of cells that have preservation of Productsurvived the manufacturing process using trypan blue exclusion.Viability Post thaw After a minimum of 2 days in LN2, the cryopreservedproduct vials are thawed and formulated in the clinical diluent. Thecells may then he centrifuged, resuspended in RPE-GM and plated ontogelatin- coated plates and cultured. The cells may be stained 2-3 weeksafter plating to mature into medium-pigmented RPE.

These assays were performed on representative cultures and preparationsof human RPE cells and confirmed that the methods described hereinyielded therapeutically useful amounts of human RPE cells that met theGTP and/or GMP standards. Further, the RPE cells described herein maycomply with at least one of the standards recited in Tables 3 or 4.Therefore, the methods described herein may be used to producetherapeutically useful amounts of human RPE cells that meet GTP and/orGMP standards for use in therapeutic applications (e.g., treatingretinal degeneration.)

Example 10 RPE Characterization and Testing

RPE cells may be seeded on gelatin. The RPE cells seeded on gelatinusually show loose pigmentation and epithelial morphology as they divideand migrate away from the initial attachment site. See, e.g.,Klimanskaya, et al. (2004) Cloning and Stem Cells 6(3):1-29, FIG. 1.However, once confluency is reestablished, the RPE cells may revert toepithelial morphology and re-expressed pigment. See, e.g., id., FIG. 2.Various tests may be performed to confirm that the RPE cells maintaintheir RPE phenotype (e.g., phenotype stability) including RPE molecularmarkers, assaying for phagocytic activity, and confirming the absence ofadventitious viruses. See, e.g., id.

RPE Molecular Markers

RPE cells express several characteristic RPE proteins in vivo, includingbestrophin, RPE65, CRALBP, and PEDF. See, e.g., id., FIG. 3. Pigmentedepithelial morphology of RPE-like differentiated derivatives of hEScells, may be lost in proliferating cultures and re-established uponreaching confluency as well as the presence of RPE molecular markersRPE65, CRALBP, bestrophin, and PEDF. Therefore, the RPE cells describedherein are similar to natural RPE cells. See also id.

Phagocytosis Assay

Functional tests for characterization of the RPE cells includeRPE-specific phagocytosis using an assay with labeled rod fragments orfluorescent S. aurelius particles. RPE cells provide functional supportto photoreceptors through phagocytosis of shed photoreceptor fragments.Therefore, phagocytosis represents a major functional characteristicidentifying RPE cells.

Approximately 500,000 formulated RPE cells may be seeded ingelatin-coated wells of a 4-well plate and cultured untilmedium-pigmentation is observed. The cells may then be incubated withfluorescent S. aurelius particles for at least about 24-36 hours atabout 37° C. A negative control may be performed with the same platesincubated at about 1-4° C. for the same duration. After the incubation,the plates may be rinsed 3 times with PBS to remove the remainingparticles, fixed with 2% paraformaldehyde in PBS, rinsed twice with PBSand examined and photographed under the fluorescence invertedmicroscope.

Human RPE cells produced according to the methods described herein arecapable of phagocytosis of both latex beads and photoreceptor fragments.

Morphological Assessment

Manually-purified, hES cell-differentiated RPE in vitro may undergosignificant morphological events in culture during the expansion phase.Single-cell suspensions plated in thin cultures depigment and cells tendto increase in surface area. The human RPE cells maintain thismorphology during expansion when the cells are rapidly dividing.However, when cell density reaches maximal capacity, RPE may take ontheir characteristic phenotypic hexagonal shape and increasepigmentation level by accumulating melanin and lipofuscin.

Routine morphological assessment may be done using a phase contrastinverted light microscope throughout the duration of the productionprocess. Digital microphotographs may be taken at key stages.Morphological assessment may be performed to confirm maintenance of theRPE phenotype. Human RPE produced according to the methods describedherein show a stable RPE phenotype, lasting over 9 months. See Example19.

Karyotyping

Karyotyping (e.g., by G-banding and FISH) may be performed to ensurethat only cells maintain a normal ploidy (e.g., 46 chromosomes forhumans). This karyotype analysis may be performed after harvest andseeding of hES cells for EB formation, after seeding of the P1 passageof RPE cells, and at the harvest of the RPE cells described herein priorto cryopreservation, for example. Human RPE produced according to themethods described herein show a stable karyotype (e.g., 46 chromosomesfor humans). See Table 1.

Adventitious Viruses

In order to confirm the absence of viral contamination RPE cells, abatch of RPE cells (RPE MA09p32) were prepared in accordance with themethods described herein. The RPE cells were passaged an additional twotimes prior to harvest and testing for viruses, to ensure that any virusis given the maximum chance to be expressed. At passage 4, RPE MA09p32+4cells were harvested and tested for inapparent viruses and in vitroviruses. A portion of the cells was passaged one further time (lot RPEMA09p32+5) before being sent for ultrastructural evaluation of viralparticles. These cells were substantially free of viral contaminationindicating that the manufacture of RPE cells does not result in hiddenviruses.

Stability Testing

To verify the RPE cells may produce the desired characteristics aftercryopreservation, vials of the RPE cells may thawed and characterized.The RPE cells may then be tested 1, 2, 3, 6, 12, 18 and 24 months postfreeze. A vial of RPE cells were prepared, cryopreserved, and thawedthen tested. These RPE cells showed a normal, 46 chromosome (XX)karyotype†, was viable, substantially free from viruses, and viableafter 6-9 months of cryostorage. Additionally, the RPE cells showed anormal, 46 chromosome (XX) karyotype†, was viable, substantially freefrom viruses, and viable after 1-4 years of cryostorage.

†These RPE cells were derived from the female human embryonic stem cellline MA09. See Klimanskaya, et al. (2007) Nat. Protoc. 2(8): 1963-72 andKlimanskaya, et al. (2006) Nature 444(7118): 481-5.

Example 11 Microarray Gene Expression Profiling of RPE Cells

A global gene expression analysis via microarray was performed on thehuman RPE cells derived from both of the single blastomere-derived hEScell lines MA01 and MA09 to test for the presence of RPE markers and theabsence of ES markers. Additionally, fetal RPE, ARPE-19, andretinoblastoma cell lines were analyzed as controls.

The data indicates that this phenotypic change to RPE is driven by achange in the global gene expression pattern of these cells,specifically with regard to the expression of PAX6, PAX2, Otx2, MitF,and Tyr. Based on ANOVA analysis comparing the respective hES cell lineto its RPE counterpart, we selected the 100 highest and lowest expressedgenes, and performed computational analysis to select genes related topluripotency and eye development. Upregulated genes are shown in Table5. Downregulated genes are shown in Table 6.

TABLE 5 Upregulated genes of interest reported on microarrays GeneSymbol Gene Name Associated with Description BEST1/VMD2 bestrophin RPEdevelopment Predominantly expressed in the basolateral (vitelliformmembrane of the retinal pigment epithelium. forms macularcalcium-sensitive chloride channels. May conduct dystrophy 2) otherphysiologically significant anions such as bicarbonate. Defects in BEST1are the cause of vitelliform macular dystrophy type 2 (VMD2); also knownas Best macular dystrophy (BMD). VMD2 is an autosomal dominant form ofmacular degeneration that usually begins in childhood or adolescence.VMD2 is characterized by typical “egg-yolk” macular lesions due toabnormal accumulation of lipofuscin within and beneath the retinalpigmented epithelium cells. Progression of the disease leads todestruction of the retinal pigmented epithelium and vision loss. Defectsin BEST1 are a cause of adult-onset vitelliform macular dystrophy(AVMD). AVMD is a rare autosomal dominant disorder with incompletepenetrance and highly variable expression. Patients usually becomesymptomatic in the fourth or fifth decade of life with a protracteddisease of decreased visual acuity. CLUL1 clusterin-like 1 retinaldevelopment Associated strongly with cone photoreceptors and (retinal)(retinal) appears in different tissues throughout retinal development.CRX cone-rod retinal development Phosphoreceptor (cone, rod) specificpaired-like homeobox homeo domain protein, expressed in developing andmature phosphoreceptor cells, binding and transactivating rhodopsin,homolog to Drosophila orthodenticle (Otx). Essential for the maintenanceof mammalian photoreceptors. CRYAA Crystallin, eye developmentCrystallins are the dominant structural components alpha A of thevertebrate eye lens. May contribute to the transparency and refractiveindex of the lens. Defects in CRYAA are the cause of zonular centralnuclear cataract one of a considerable number of phenotypically andgenotypically distinct forms of autosomal dominant cataract. Thiscongenital cataract is a common major abnormality of the eye thatfrequently causes blindness in infants. Crystallins do not turn over asthe lens ages, providing ample opportunity for post-translationalmodifications or oxidations. These modifications may change crystallinsolubility properties and favor senile cataract. CRYBA1 crystallin, betaeye development Crystallins are the dominant structural components A1 ofthe vertebrate eye lens. Crystallins do not turn over as the lens ages,providing ample opportunity for post-translational modifications oroxidations. These modifications may change crystallin solubilityproperties and favor senile cataract. CRYBA2 crystallin, beta eyedevelopment Crystallins are the dominant structural components A2 of thevertebrate eye lens. Crystallins do not turn over as the lens ages,providing ample opportunity for post-translational modifications oroxidations. These modifications may change crystallin solubilityproperties and favor senile cataract. CRYBA4 crystallin, beta eyeCrystallins are the dominant structural components A4 development of thevertebrate eye lens. Defects in CRYBA4 are the cause of lamellarcataract 2. Cataracts are a leading cause of blindness worldwide,affecting all societies. A significant proportion of cases aregenetically determined. More than 15 genes for cataracts have beenidentified, of which the crystallin genes are the most commonly mutated.Lamellar cataract 2 is an autosomal dominant congenital cataract.Defects in CRYBA4 are a cause of isolated microphthalmia with cataract 4(MCOPCT4). Microphtalmia consists of a development defect causingmoderate or severe reduction in size of the eye. Opacities of the corneaand lens, scaring of the retina and choroid, and other abnormalitieslike cataract may also be present Crystallins do not turn over as thelens ages, providing ample opportunity for post-translationalmodifications or oxidations. These modifications may change crystallinsolubility properties and favor senile cataract. CRYBB1 crystallin, betaeye development Crystallins are the dominant structural components B1 ofthe vertebrate eye lens. CRYBB2 crystallin, beta eye developmentCrystallins are the dominant structural components of B2 the vertebrateeye lens. Defects in CRYBB2 are the cause of congenital ceruleancataract 2 (CCA2); also known as congenital cataract blue dot type 2.CCA2 is a form of autosomal dominant congenital cataract (ADCC).Cerulean cataracts have peripheral bluish and white opacifications inconcentric layers with occasional central lesions arranged radially.Although the opacities may be observed during fetal development andchildhood, usually visual acuity is only mildly reduced until adulthood,when lens extraction is generally necessary. Defects in CRYBB2 are thecause of sutural cataract with punctate and cerulean opacities (CSPC).The phenotype associated with this form of autosomal dominant congenitalcataract differed from all other forms of cataract reported. Defects inCRYBB2 are a cause of Coppock-like cataract (CCL). Crystallins do notturn over as the lens ages, providing ample opportunity for post-translational modifications or oxidations. CRYBB3 crystallin, beta eyedevelopment Crystallins are the dominant structural components B3 of thevertebrate eye lens. Defects in CRYBB3 are the cause of autosomalrecessive congenital nuclear cataract 2 (CATCN2); a form of non-syndromic congenital cataract. Non-syndromic congenital cataracts varymarkedly in severity and morphology, affecting the nuclear, cortical,polar, or subcapsular parts of the lens or, in severe cases, the entirelens, with a variety of types of opacity. They are one of the majorcauses of vision loss in children worldwide and are responsible forabout one third of blindness in infants. Congenital cataracts can leadto permanent blindness by interfering with the sharp focus of light onthe retina during critical developmental intervals. Crystallins do notturn over as the lens ages, providing ample opportunity forpost-translational modifications or oxidations. These modifications maychange crystallin solubility properties and favor senile cataract.DCT/TYRP2 dopachrome pigmented cells Tyrosine metabolism and Melaninbiosynthesis. tautomerase (dopachrome delta- isomerase, tyrosine-related protein 2) LHX2 LIM development/ Transcriptional regulatoryprotein involved in the homeobox 2 differentiation control of celldifferentiation in developing lymphoid and neural cell types. LIM2 lensintrinsic eye Present in the thicker 16-17 nm junctions of membranedevelopment mammalian lens fiber cells, where it may contribute toprotein 2, cell junctional organization. Acts as a receptor for 19kDacalmodulin. May play an important role in both lens development andcataractogenesis. MITF microphthalmia- RPE Transcription factor fortyrosinase and tyrosinase- associated development related protein 1.Binds to a symmetrical DNA transcription sequence (E-boxes)(5′-CACGTG-3′) found in the factor tyrosinase promoter. Plays a criticalrole in the differentiation of various cell types as neural crest-derived melanocytes, mast cells, osteoclasts and optic cup-derivedretinal pigmented epithelium. Highly expressed in retinal pigmentedepithelium. OCA2 oculocutaneous pigmented cells Could be involved in thetransport of tyrosine, albinism II the precursor to melanin synthesis,within the (pink-eye melanocyte. Regulates the pH of melanosome anddilution the melanosome maturation. One of the homolog, components ofthe mammalian pigmentary system. mouse) Seems to regulate thepostranslational processing of tyrosinase, which catalyzes the limitingreaction in melanin synthesis. May serve as a key control point at whichethnic skin color variation is determined. Major determinant of brownand/or blue eye color. Defects in OCA2 are the cause of oculocutaneousalbinism type II (OCA2). OCA2 is an autosomal recessive form ofalbinism, a disorder of pigmentation in the skin, hair, and eyes. Thephenotype of patients with OCA2 is typically somewhat less severe thanin those with tyrosinase- deficient OCA1 . There are several forms ofOCA2, from typical OCA to relatively mild ‘autosomal recessive ocularalbinism’ (AROA). OCA2 is the most prevalent type of albinism throughoutthe world. The gene OCA2 is localized to chromosome 15 at 15q 11.2-q12OPN3 opsin 3 eye May play a role in encephalic photoreception. Stronglydevelopment expressed in brain. Highly expressed in the preoptic areaand paraventricular nucleus of the hypothalamus. Shows highly patternedexpression in other regions of the brain, being enriched in selectedregions of the cerebral cortex, cerebellar Purkinje cells, a subset ofstriatal neurons, selected thalamic nuclei, and a subset of interneuronsin the ventral horn of the spinal cord. OPN5 opsin 5 eye Associated withvisual perception and phototransduction. development OTX2 orthodenticleretinal Probably plays a role in the development of the homolog 2development brain and the sense organs. Defects in OTX2 are the(Drosophila) cause of syndromic microphthalmia 5 (MCOPS5).Microphthalmia is a clinically heterogeneous disorder of eye formation,ranging from small size of a single eye to complete bilateral absence ofocular tissues. Up to 80% of cases of microphthalmia occur inassociation with syndromes that include non-ocular abnormalities such ascardiac defects, facial clefts, microcephaly and hydrocephaly. MCOPS5patients manifest unilateral or bilateral microphthalmia/clinicalanophthalmia and variable additional features including coloboma,microcornea, cataract, retinal dystrophy, hypoplasia or agenesis of theoptic nerve, agenesis of the corpus callosum, developmental delay, jointlaxity, hypotonia, and seizures. PAX6 paired box RPE Transcriptionfactor with important functions in the gene 6 development development ofthe eye, nose, central nervous (aniridia, system and pancreas. Requiredfor the differentiation keratitis) of pancreatic islet alpha cells (Bysimilarity). Competes with PAX4 in binding to a common element in theglucagon, insulin and somatostatin promoters (By similarity). Isoform 5aappears to function as a molecular switch that specifies target genes.Defects in PAX6 results in a number of eye defects and malformations.PHC2 polyhomeotic- development/ Component of the Polycomb group (PcG)like 2 differentiation multiprotein PRC1 complex, a complex required to(Drosophila) maintain the transcriptionally repressive state of manygenes, including Hox genes, throughout development. PeG PRC1 complexacts via chromatin remodeling and modification of histones: it mediatesmonoubiquitination of histone H2A ‘Lys-119’, rendering chromatinheritably changed in its expressibility. PKNOX2 PBX/knotted 1development/ Known to be involved in development and may, homeobox 2differentiation along with MEIS, control PAX6. PRKCA protein kinasecellular signaling Very important for cellular signaling pathways suchC, alpha as the MAPK, Wnt, P13, VEGF and Calcium pathways. PROX1prospero- eye May play a fundamental role in early development relateddevelopment of CNS. May regulate gene expression and homeobox 1development of postmitotic undifferentiated young neurons. Highlyexpressed in lens, retina, and pancreas. PRRX1 paired relateddevelopment/ Necessary for development. Transcription coactivator,homeobox 1 differentiation enhancing the DNA-binding activity of serumresponse factor. RAI1 retinoic acid development/ May function as atranscriptional regulator. Regulates induced 1 differentiationtranscription through chromatin remodeling by interacting with otherproteins in chromatin as well as proteins in the basic transcriptionalmachinery. May be important for embryonic and postnatal development. Maybe involved in neuronal differentiation. RARA retinoic acid development/This is a receptor for retinoic acid. This metabolite receptor, alphadifferentiation has profound effects on vertebrate development. Thisreceptor controls cell function by directly regulating gene expression.RARB retinoic acid development/ This is a receptor for retinoic acid.This metabolite receptor, beta differentiation has profound effects onvertebrate development. This receptor controls cell function by directlyregulating gene expression. RARRES1 retinoic acid development/Associated with differentiation and control of cell receptordifferentiation proliferation. May be a growth regulator that respondermediates some of the growth suppressive effects of (tazaroteneretinoids. induced) 1 RAX retina and eye Plays a critical role in eyeformation by regulating anterior neural development the initialspecification of retinal cells and/or their fold homeobox subsequentproliferation. Binds to the photoreceptor conserved element-I(PCE-1/Ret 1) in the photoreceptor cell-specific arrestin promoter. RB1retinoblastoma development/ An important regulator of other genes andcell growth. 1 (including differentiation Defects in RBI are the causeof childhood osteosarcoma) cancer retinoblastoma (RB). RB is acongenital malignant tumor that arises from the nuclear layers of theretina. RDH5 retinol RPE retinol dehydrogenase 5, 11-cis, expressed inretinal dehydrogenase development pigmented epithelium, formerly RDH 1.5 (11-cis/9-cis) Stereospecific 11-cis retinol dehydrogenase, whichcatalyzes the final step in the biosynthesis of 11-cis retinaldehyde,the universal chromophore of visual pigments: Abundant in the retinalpigmented epithelium. Defects in RDH5 are a cause of fundusalbipunctatus (FA). FA is a rare form of stationary night blindnesscharacterized by a delay in the regeneration of cone and rodphotopigments. RGR retinal G RPE Preferentially expressed at high levelsin the retinal protein development pigmented epithelium (RPE) andMueller cells of coupled the neural retina. Retinal opsin related,(rhodopsin receptor homolog)expressed in the retinal pigmentedepithelium, encoding a retinaldehyde, preferentially all-trans retinal,binding protein, G protein coupled receptor superfamily. RLBP1/retinaldehyde RPE Carries I 1-cis-retinol and 11-cis-retinaldehyde asCRALBP1 binding development endogenous ligands and may be a functionalprotein 1 component of the visual cycle. Defects in RLBP1 are a cause ofautosomal recessive retinitis pigmentosa (arRP). Retinitis pigmentosa(RP) leads to degeneration of retinal photoreceptor cells. dystrophy,also known as Vasterbotten dystrophy. It is another form of autosomalrecessive retinitis pigmentosa. Defects in RLBP1 are the cause ofNewfoundland rod-cone dystrophy (NFRCD). NFRCD is a retinal dystrophyreminiscent of retinitis punctata albescens but with a substantiallylower age at onset and more-rapid and distinctive progression. RPE65retinal pigment RPE Retinal pigmented epithelium specific. Retinalepithelium- development pigmented epithelium-specific 65, majorrnicrosomal specific protein, minor role in the isomerisation ofall-trans to protein 65kDa 11-cis retinal, associated with theendoplasmic reticulum, also expressed in renal tumor cells. Playsimportant roles in the production of 11-cis retinal and in visualpigment regeneration. RRH retinal pigment RPE Found only in the eye,where it is localized to the epithelium- development retinal pigmentepithelium (RPE). In the RPE, it is derived localized to the microvillithat surround the rhodopsin photoreceptor outer segments. May play arole in rpe homolog physiology either by detecting light directly or bymonitoring the concentration of retinoids or other photoreceptor-derivedcompounds. RTN 1 reticulon 1 development/ Expressed in neural andneuroendocrine tissues differentiation and cell cultures derivedtherefrom. Expression of isoform RTN1-C is strongly correlated withneuronal differentiation. RXRB retinoid X development/ Nuclear hormonereceptor. Involved in the retinoic receptor, beta differentiation acidresponse pathway. Binds 9-cis retinoic acid (9C-RA), obligate member ofheterodimeric nuclear receptors. steroid/thyroid/retinoic receptorsuperfamily. RXRG retinoid X development/ Nuclear hormone receptor.Involved in the retinoic receptor, differentiation acid responsepathway. Binds 9-cis retinoic acid gamma (9C-RA), obligate member ofheterodimeric nuclear receptors. steroid/thyroid/retinoic receptorsuperfamily. SERPINF1/ serpin RPE Specific expression in retinal pigmentepithelial PEDF peptidase development cells and blood plasma.Neurotrophic protein; inhibitor, clade induces extensive neuronaldifferentiation F (alpha-2 in retinoblastoma cells. antiplasmin, pigmentepithelium derived factor), member 1 SIX3 sine oculis eye Expressedduring eye development in midline homeobox development forebrain and inanterior region of the neural plate homolog 3 especially inner retinaand later in ganglion cells (Drosophila) and in cells of the innernuclear layer, involved in regulation of eye development. SOX10 SRY (sexdevelopment/ Transcription factor that seems to function determiningdifferentiation synergistically with other development associated regionY)-box 10 proteins. Could confer cell specificity to the function ofother transcription factors in developing and mature glia. SOX5 SRY (sexdevelopment/ Expression is associated with craniofacial, determiningdifferentiation skeletal and cartilage development and is highlyexpressed region Y)-box 5 in brain, testis and various tissues. SOX6 SRY(sex development/ Expression is associated with craniofacial, skeletaldetermining differentiation and cartilage development and is highlyexpressed region Y)-box 6 in brain, testis and various tissues. SOX8 SRY(sex development/ May play a role in central nervous system, determiningdifferentiation limb and facial development region Y)-box 8 SOX9 SRY(sex development/ Plays an important role in the normal development.determining differentiation May regulate the expression of other genesregion Y)-box 9 involved for skeletal and cartilage formation by(campomelic acting as a transcription factor for these genes. dysplasia,autosomal sex- reversal) TIMP3 TIMP RPE Matrix metalloproteinase, tissueinhibitor 3, metallopeptidase development expressed in retinal pigmentepithelium, placenta, inhibitor 3 localized in extracellular matrix.Complexes with (Sorsby fundus metalloproteinases (such as collagenases)and dystrophy, irreversibly inactivates them. May form part of apseudoinflam tissue-specific acute response to remodeling matory)stimuli. Defects in TIMP3 are the cause of Sorsby fundus dystrophy(SFD). SFD is a rare autosomal dominant macular disorder with an age ofonset in the fourth decade. It is characterized by loss of centralvision from subretinal neovascularization and atrophy of the oculartissues. TTR transthyretin (prealbumin, Thyroid hormone-binding protein.Probably transports amyloidosis type I) thyroxine from the bloodstreamto the brain. Defects in TTR are the cause of amyloidosis VII; alsoknown as leptomeningeal amyloidosis or meningocerebrovascularamyloidosis. Leptomeningeal amyloidosis is distinct from other forms oftransthyretin amyloidosis in that it exhibits primary involvement of thecentral nervous system. Neuropathologic examination shows amyloid in thewalls of leptomeningeal vessels, in pia arachnoid, and subpial deposits.Some patients also develop vitreous amyloid deposition that leads tovisual impairment (oculoleptomeningeal amyloidosis). TYR tyrosinasepigmented cells This is a copper-containing oxidase that functions in(oculocutaneo the formation of pigments such as melanins and us albinismother polyphenolic compounds. Defects in TYR 1A) are the cause ofoculocutaneous albinism type IA (OCA-IA). OCA-I, also known astyrosinase negative oculocutaneous albinism, is an autosomal recessivedisorder characterized by absence of pigment in hair, skin and eyes.OCA-I is divided into 2 types: type IA, characterized by complete lackof tyrosinase activity due to production of an inactive enzyme, and typeIB characterized by reduced activity of tyrosinase. OCA-IA patientspresents with the lifelong absence of melanin pigment after birth andmanifest increased sensitivity to ultraviolet radiation and topredisposition to skin cancer defects in TYR are the cause ofoculocutaneous albinism type IB (OCA-IB); also known as albinism yellowmutant type. OCA-IB patients have white hair at birth that rapidly turnsyellow or blond. TYRP1 tyrosinase- pigmented cells Specific expressionin Pigment cells. Oxidation of related protein 15,6-dihydroxyindole-2-carboxylic acid (DHICA) into indole-5,6-quinone-2-carboxylic acid. May regulate or influence the type ofmelanin synthesized. Defects in TYRP1 are the cause of rufousoculocutaneous albinism (ROCA). ROCA occurs in blacks and ischaracterized by bright copper-red coloration of the skin and hair anddilution of the color of the iris. Defects in TYRP1 are the cause ofoculocutaneous albinism type III (OCA-III); also known as OCA3. OCA-IIIis a form of albinism with only moderate reduction of pigment.Individuals with OCA-III are recognized by their reddish skin and haircolor.

TABLE 6 Downregulated genes of interest reported on microarrays GeneSymbol Gene Name Associated with Description ALPL alkaline ES cellsElevated expression of this enzyme is associated phosphatase withundifferentiated pluripotent stem cell. CECR2 cat eye Part of the CERF(CECR2-containing-remodeling syndrome factor) complex, which facilitatesthe perturbation of chromosome chromatin structure in an ATP-dependentmanner. region, May be involved through its interaction with candidate 2LRPPRC in the integration of cytoskeletal network with vesiculartrafficking, nucleocytosolic shuttling, transcription, chromosomeremodeling and cytokinesis. Developmental disorders are associated withthe duplication of the gene. DCAMKL1 doublecortin Embryonic Probablekinase that may be involved in a calcium- and CaM development signalingpathway controlling neuronal migration in kinase-like 1 the developingbrain. DPPA2 developmental ES cells May play a role in maintaining cellpluripotency pluripotentiality. associated 2 DPPA3 developmental EScells May play a role in maintaining cell pluripotentiality.pluripotency associated 3 DPPA4 developmental ES cells May indicate cellpluripotentiality. pluripotency associated 4 DPPA5/Esg1 developmental EScells Embryonic stem cell marker. pluripotency associated 5/Embryonicstem cell specific gene 1 FOXD3 forkhead box Pluripotence Required formaintenance of pluripotent stem cells D3 in the pre-implantation andperi-implantation stages of embryogenesis. LITDIECAT11 LINE-1 type EScells Embryonic stem cell marker. transposase domain containing 1/EScell associated transcript 11 NANOG NANOG ES cells Embryonic stem cellmarker. Transcription regulator homeobox involved in inner cell mass andembryonic stem (ES) cells proliferation and self-renewal. Imposespluripotency on ES cells and prevents their differentiation towardsextraembryonic endoderm and trophectoderm lineages. NCAM1 neural cellneuroprogenitors This protein is a cell adhesion molecule involved inadhesion neuron-neuron adhesion, neurite fasciculation, molecula 1outgrowth of neurites, etc. NES/Nestin nestin ES cells Neuralprogenitorcells. NODAL nodal Embryonic Essential for mesoderm formation and axialpatterning development during embryonic development. NR5A2/FTF nuclearEmbryonic May contribute to the development and regulation of receptordevelopment liver and pancreas-specific genes and play subfamily 5,important roles in embryonic development. group A, member 2 POU5Fl/ POUdomain, ES cells Embryonic stem cell marker. Indicator of “Sternness”.Oct-3/4 class 5, Transcription factor that binds to the octamertranscription motif (5′-ATITGCAT-3′). Prime candidate for an factor 1early developmental control gene. SOX17 SRY (sex Inhibitor of Negativeregulator of the Wnt signaling pathway. determining differentiationregion Y)-box 17 SOX2 SRY (sex ES cells Indicator of “Sternness”.Expressed in inner cell determining mass, primitive ectoderm anddeveloping CNS. region Y)-box 2 TBX3 T-box 3 (ulnar EmbryonicTranscriptional repressor involved in developmental mammary developmentprocesses. Murine T-box gene Tbx3 (T, syndrome) brachyury) homolog,putative transcription factor, pairing with TBX5, homolog to Drosophilaoptomotor-blind gene (omb), involved in optic lobe and wing development,involved in developmental regulation, expressed in anterior andposterior mouse limb buds, widely expressed in adults TDGFl/Cripto-1teratocarci- ES cells Indicator of “Sternness”. Could play a role in thenoma-derived determination of the epiblastic cells that subsequentlygrowth factor 1 give rise to the mesoderm. TEK/VMCM TEK tyrosine EarlyThis protein is a protein tyrosine-kinase transmembrane kinase,Endothelial receptor for angiopoietin 1. It may constitute endothelialprogenitors the earliest mammalian endothelial cell lineage marker.(venous Probably regulates endothelial cell malformations,proliferation, differentiation and guides the proper multiple patterningof endothelial cells during blood vessel cutaneous and formationmucosal) TUBB2A, tubulin, beta neuroprogenitors Tubulin is the majorconstituent of microtubules. TUBB2B 2A, tubulin, It binds two moles ofGTP, one at an exchangeable beta 2B site on the beta chain and one at anon-exchangeable site on the alpha-chain. Often associated with theformation of gap junctions in neural cells. TUBB2A, tubulin, betaneuroprogenitors Tubulin is the major constituent of microtubules. ItTUBB2B, 2A, tubulin, binds two moles of GTP, one at an exchangeableTUBB2C, beta 2B, site on the beta chain and one at a non-exchangeableTUBB3, tubulin, beta site on the alpha-chain. Often associated with theTUBB4 2C, tubulin, formation of gap junctions in neural cells. beta 3,tubulin, beta 4 TUBB3 tubulin, beta 3 neuroprogenitors Tubulin is themajor constituent of microtubules. It hinds two moles of GTP, one at anexchangeable site on the beta chain and one at a non-exchangeable siteon the alpha-chain. Often associated with the formation of gap junctionsin neural cells. TWIST1 twist homolog 1 Inhibitor of Probabletranscription factor, which negatively differentiation regulatescellular determination and differentiation. UTF1 undifferentiated EScells Embryonic stem cell marker. Acts as a embryonic transcriptionalcoactivator of ATF2. cell transcription factor 1 VSNL1 visinin-like 1Inhibitor of Regulates the inhibition of rhodopsin phosphorylation.rhodopsin ZFP42/Rex-1 zinc finger ES cells Embryonic Stem cell marker.protein 42

The results of the microarray assay demonstrates that RPE cells made bythe methods described herein express multiple genes that are notexpressed by hES cells, fetal RPE cells, or ARPE-19 cells. Thedistinctive molecular fingerprint of mRNA and protein expression in theES-cell derived RPE cells of the invention constitutes a set of markers,such as RPE-65, Bestrophin, PEDF, CRABLP, Otx2, Mit-F, PAX6 and PAX2,that make these RPE cells distinct from cells in the art, such as hEScells, ARPE-19 cells, and fetal RPE cells.

Example 12 RPE-Specific mRNA Expression Measured by Quantitative,Real-Time, Reverse Transcription PCR (QPCR)

In order to characterize developmental stages during the human embryonicstem cell (hES) differentiation process into retinal pigmentedepithelium (RPE) assays were employed to identify the expression levelsof genes key to each representative stage of development. qPCR wasdeveloped to provide a quantitative and relative measurement of theabundance of cell type-specific mRNA transcripts of interest in the RPEdifferentiation process. qPCR was used to determine genes that areexpressed in human embryonic stem cells, in neuroretinal cells duringeye development, and in RPE cells differentiated from human embryonicstem cells. The genes for each cell type are listed below in Table 7.

TABLE 7 Genes specific to hES, neuroretina/eye, and RPE cells ESCell-Specific Neuroectoderm /Neuroretina RPE-Specific Genes Oct-4(POU5F1) Chx 10 PAX-6 NANOG NCAM PAX2 Rex-1 Nestin RPE-65 TDGF-1β-Tubulin PEDF SOX-2 CRALBP DPPA-2 Bestrophin MitF Otx-2 Tyr

It was determined that hES-specific genes included Oct-4 (POU5F1),NANOG, Rex-1, TDGF-1, SOX-2, and DPPA-2. Genes specific to neuralectoderm/neural retina include CHXIO, NCAM, Nestin, and β-Tubulin. Bycontrast, RPE cells differentiated from human embryonic stem cells werefound to express PAX-6, PAX2, RPE-65, PEDF, CRALBP, Bestrophin, MitF,Otx-2, and Tyr by qPCR measurement.

As evident from the qPCR tests, hES-specific genes are grosslydownregulated (near 1000-fold) in RPE cells derived from hES, whereasgenes specific for RPE and neuroectoderm are vastly upregulated (about100-fold) in RPE cells derived from hES. In addition, qPCR analysis offully mature RPE demonstrated a high level expression of theRPE-specific markers RPE65, Tyrosinase, PEDF, Bestrophin, MitF, andPAX6. This agrees with current literature regarding the Pax2-inducedregulation of MitF and downstream activation of genes associated withterminally differentiated RPE.

The results of the assay demonstrates that RPE cells made by the methodsdescribed herein express multiple genes at the mRNA level that are notexpressed by hES cells or neural ectoderm/neural retina cells. Thus thedistinctive molecular fingerprint of mRNA in the ES-cell derived RPEcells of the invention constitutes a set of markers, such as RPE-65,tyrosinase, Bestrophin, PEDF, Mit-F, and PAX6, that make these RPE cellsdistinct from cells in the art, such as hES cells and neuralectoderm/neural retina cells. This assay also confirms that the humanRPE cell preparations made in accordance with the methods describedherein are substantially free from hES cell contamination.

Example 13 RPE-Specific Protein Expression Identified by Western BlotAnalysis

To identify proteins expressed in the human RPE cells, a subset ofhES-specific and RPE-specific markers were assayed by Western Blot.Actin was used as protein loading control.

The Western blot analysis confirms that the human RPE cells derived fromhES cells did not express the hES-specific proteins Oct-4, NANOG, andRex-1, whereas they expressed RPE65, CRALBP, PEDF, Bestrophin, PAX6, andOtx2. These proteins are therefore prominent markers of RPE cellsdifferentiated from hES cells. By contrast, APRE-19 cells showed aninconclusive pattern of proteomic marker expression. See WO 2009/051671,FIG. 6.

The results of the assay demonstrates that RPE cells made by the methodsdescribed herein express multiple genes at the protein level that arenot expressed by hES cells or APRE-19 cells. Thus the distinctivemolecular fingerprint of protein expression in the ES-cell derived RPEcells of the invention constitutes a set of markers, such as RPE65,CRALBP, PEDF, Bestrophin, PAX6, and Otx2, that make these RPE cellsdistinct from cells in the art, such as hES cells and APRE-19 cells.This assay also confirms that the human RPE cell preparations made inaccordance with the methods described herein are substantially free fromhES cell contamination.

Example 14 Cryopreserved Preparations of Human RPE Cells

It is preferable that human RPE cells require reach a level of mediumpigmentation prior to cryopreservation. PCR may used to determine if thecells are ready for cryopreservation (e.g., appropriate levels of RPEspecific markers). Seven lots of human RPE cells (090621, 090606,1211606, AB3, A090609, A090714, and A020101R04) manufactured assayed forthe selected hES and RPE markers.

For each lot, qRT-PCR assays for the seven markers were conducted intriplicate on at least 2 and up to 5 separate days. Data were normalizedto the β-actin expression observed in each sample during each run andcompared to the level of expression in the MA09 hES reference, alsodetermined in each experimental run (MA09 hES cells were used as thepluripotent stem cells in the methods to make these seven lots of humanRPE cells). For each of the seven lots, the mean expression for eachmarker was then calculated. To give each lot equal weight, the mean ofthe means for the seven RPE lots was then determined. Individual RPE lotmeans and the collective means for the four RPE markers and the threehES markers are shown in Table 8. Also shown are the highest and thelowest individual observed value for each of the markers. The datapresented in Table 8 were plotted in the bar graph depicted in FIG. 8.

TABLE 8 RPE Gene Expression Relative to MA09 hES Mean Log 10 Mean Log 10Upregulation Downregulation RPE Cells Markers hES Cell Markers RPE LotRPE65 PAX6 BESTROPHIN MITF OCT4 NANOG SOX2 090621 3.55 1.99 3.66 2.48−3.05 −2.87 −1.74 (n = 4) (n = 5) (n = 5) (n = 5) (n = 5) (n = 3) (n =5) 090606 2.06 1.89 2.03 2.20 −2.87 −2.92 −1.54 (n = 2) (n = 2) (n = 2)(n = 2) (n = 2) (n = 2) (n = 2) 1211606 2.63 2.24 2.53 2.22 −2.68 −2.63−1.19 (n = 2) (n = 2) (n = 2) (n = 2) (n = 2) (n = 2) (n = 2) AB3 3.432.14 3.94 2.24 −2.83 −2.87 −2.20 (n = 3) (n = 4) (n = 4) (n = 5) (n = 4)(n = 3) (n = 4) A090609 3.18 1.95 3.47 2.42 −3.17 −2.44 −2.26 (n = 4) (n= 4) (n = 5) (n = 5) (n = 5) (n = 4) (n = 4) A090714 3.76 2.06 4.05 2.78−3.13 −2.44 −1.80 (n = 4) (n = 4) (n = 5) (n = 5) (n = 5) (n = 4) (n =5) A020101R04 2.33 1.95 3.35 2.00 −3.45 −2.93 −2.28 (n = 3) (n = 3) (n =4) (n = 5) (n = 4) (n = 3) (n = 4) MEAN 2.99 2.03 3.29 2.33 −3.03 −2.73−1.86 of MEANS (n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7)SD 0.65 0.12 0.74 0.25   0.26   0.22   0.41 Low 1.80 1.30 1.59 1.80−2.56 −2.10 −1.14 High 4.50 3.00 4.50 3.50 −3.70 −3.30 −2.60

The results of the assay demonstrates that human RPE cells made by themethods described herein express multiple genes that are not expressedby hES cells. Thus the distinctive molecular fingerprint of proteinexpression in the ES-cell derived RPE cells of the invention constitutesa set of markers, such as RPE65, CRALBP, PEDF, Bestrophin, PAX6, andOtx2, that make these RPE cells distinct from hES cells. Accordingly,the human RPE cells described herein show upregulation of the RPE cellmarkers, RPE65, PAX6, bestrophin, and MITE, and downregulation of the EScell markers, OCT4, NANOG, and SOX2, confirming that the human RPE cellsare fully differentiated and have lost their pluripotency. This assayalso confirms that the human RPE cell preparations are substantiallyfree from hES cell contamination. Further, these RPE cells are at adesirable level of pigmentation so that they may be cryopreserved andthawed with high levels of viability after thawing.

Example 15 Pharmaceutical Preparations of Human RPE Cells

Manufacture of Pharmaceutical Preparations of human RPE

Pharmaceutical preparations of human RPE cells may be manufacturedaseptically in a Class 100 biological safety cabinet. The diluentutilized for the pharmaceutical preparations may be ALCON BSS Plus®Intraocular Irrigating Solution, a sterile balanced salt solution,comprising sodium chloride (NaCl) 7.14 mg, potassium chloride (KCl) 0.38mg, calcium chloride dihydrate (CaCl₂*H₂O) 0.154 mg, magnesium chloridehexahydrate (MgCl₂*6H₂O) 0.2 mg, dibasic sodium phosphate (HNa₂PO₄) 0.42mg, sodium bicarbonate (NaHCO₃) 2.1 mg, dextrose 0.92 mg, glutathionedisulfide 0.184 mg, and sodium hydroxide and/or hydrochloric acid toadjust pH and water for injection per milliliter (mL). The pH is about7.5 and the osmolality about 305 mOsm/Kg.

Prior to injection, the RPE cells may be thawed for use. The vial ofcells may be removed from the liquid nitrogen freezer, placed in a waterbath at 37° C., and constantly agitated until the entire contents areliquid. For each cryovial, the thawed contents may be resuspended in 1mL of RPE-MM and transferred to a separate sterile 50 mL tube. RPE-MMare added to each conical tube to bring the volume to 40 mL. The tubemay then centrifuged, the supernatant aspirated and the pelletresuspended in 40 mL of BSS-Plus. The cell suspension may be againcentrifuged, the supernatant aspirated. The pellet may be resuspended ina second volume of 40 mL of BSS-Plus and the cells pelleted bycentrifugation a third time.

The resulting pellet may be resuspended in about 75 μL of BSS-Plus pervial thawed and the cells transferred to a sterile 0.5 mL sterilemicrocentrifuge tube. A viable cell count may be performed and theappropriate volume of BSS-Plus is added to achieve the appropriatedensity of cells for dosing. The pharmaceutical preparations of humanRPE cells may have a preparation viability of at least about 85%. Thesecells may maintain this viability for at least about 4 hours postpreparation. A 200 μL sample of the formulated product may be placed ina sterile microcentrifuge tube. The vial may be placed on ice fortransport to the surgical facility and is stable for at least about 4hours after preparation (e.g., cells may be used in therapy within atleast about 4 hours of preparation). See FIG. 7.

DMSO Levels in Pharmaceutical Preparation

Three exemplary lots of RPE cells: 090621, MA09p334+2, and MA01p50+4.Each lot was thawed and a final dose preparation was prepared asdescribed herein to achieve a cell density of 1333 viable cells/μL(e.g., equivalent to about a 2×10⁵ cell dose). A 200 μL sample of thecell suspension was transferred to cryovials, frozen at −20° C. andshipped to a testing lab for determination of DMSO residual levels usinggas chromatography.

The results indicate that preparation of the pharmaceutical preparationof RPE lots 090621, MA09p334+2, and MA01p50+4 resulted in extremely lowDMSO residual levels (ppm) (e.g., below levels considered acceptable forclinical administration). Therefore the preparation of the RPE cellsdescribed results in DMSO residual levels acceptable for clinicaladministration.

Endotoxin Levels in Pharmaceutical Preparation

Three exemplary lots of RPE cells: 090621, MA09p334+2, and MA01p50+4.Each lot was thawed and a final dose preparation was prepared aspreviously described to achieve a cell density of 1333 viable cells/μL(e.g., equivalent to about a 2×10⁵ cell dose). A 100 μL sample of thecell suspension was transferred to cryovials, stored at 4° C. andshipped to a testing lab for endotoxin levels using a kineticturbidimetric assay with a sensitivity of 0.001 EU/mL.

The results indicate that preparation of the clinical formulation usingRPE lots 090621, MA09p34+2, and MA01p50+4 resulted in endotoxin levelsof <0.100, 0.993 and <0.100 EU/mL, respectively. Therefore thepreparation of the RPE cells according to the methods described hereinresults in endotoxin levels acceptable for clinical administration. Thusthe human RPE cells prepared according to the methods described hereinmay be prepared, stored, thawed, and formulated in a pharmaceuticalpreparation suitable for therapeutic applications.

Example 16 Capillary and Cannula Cell Delivery Systems

Needle/syringe and cannula systems were tested for damage/loss of humanRPE cells (e.g., cell viability/activity, cell adhesion to the syringe)at a cell dose of about 1×10⁵ human RPE cells in a small volume (e.g.,about 2-3 μL).

Capillary Cell Delivery System

Cryopreserved vials of RPE lot 090621 were thawed and formulated in apharmaceutical preparation. The resulting RPE were formulated inBSS-Plus and resuspended at 50,000 viable cells per microliter (μL).

The capillary delivery system used was a 25 μL Hamilton syringe and astandard glass capillaries made by World Precision Instruments (WPI),Standard Glass Capillaries: 4 in. (100 mm); 1.5/0.84 OD/ID (mm)filament, fire polished using natural gas.

The Hamilton syringe and glass capillaries were autoclaved prior to use.The tubing was flushed with 70% sterile ethanol using a syringe andneedle. This was followed by thorough flushing with sterile PBS prior touse. A 20 gauge syringe needle was affixed to the syringe. One end ofthe tubing was fitted to the needle and the other end of the tubing wasinserted over the capillary tube.

BSS-Plus was drawn into the capillary, tubing, and syringe. BSS-Plus wasthen expelled until about 2-3 inches of the tubing was void to ensurethat there was an air bubble between the cells and medium. About 10-12μL of the cell suspension was drawn into the capillary. About 2 μLs ofthe cell suspension was dispensed over about a 10-20 second timeinterval into a sterile microcentrifuge tube. The dispensing wasrepeated 8 times until about 16 μl had been delivered over about a 1.5-2minute period.

The RPE cells were then assessed for assessed for viable cell number andtheir ability to grow in culture. Samples of RPE cells that had beendelivered through the capillary were tested for viable cell number bytrypan blue exclusion and compared to the same formulated RRE cells thathad not been delivered. Control and capillary subjected cells were alsoseeded in 4-well plates at 50,000 viable cells per well in 1 mL of RPEgrowth medium. After four days in culture, control and capillarydelivered RPE cells were trypsinized and cell counts were performed.

Vials of cryopreserved RPE cells (lot 090621) were thawed, washed andresuspended in BSS-Plus at a concentration of 50,000 viable cells permicroliter. The viability of the formulated RPE was 88%. The viable cellcounts performed on RPE preparation that had been delivered through thecapillary system versus control cells are shown in the Table 9.

TABLE 9 Viable Cell Counts Control Capillary Injected RPE Cells RPECells Viable Cell/μL 50,500 47,875 Percent Viability 92 93 ViableCells/Well 283,125 279,375 (seeded at about 50,000 cells)

To assess longer-term survival, aliquots of RPE capillary delivered andcontrol RPE cells were seeded at 50,000 viable cells per well andcultured for four day before harvesting and counting. These results arealso shown in Table 8.

Capillary-injected and non-injected RPE showed no difference regardingthe viability, viable cell number or the ability to propagate inculture. The capillary-injection system used in the preclinical studieshad no adverse effects RPE number, viability or their capacity toproliferate in culture.

Cannula Cell Delivery System

A study was done to confirm that the use of the cell delivery system, a30-gauge Angled Rigid Injection Cannula, (Synergetics Inc.), does nothave an impact on the viability or survivability of the RPE cells. Thisstudy was performed with nominal cell concentrations of 800 cells/μL and1,000 cells/μL.

Cryopreserved RPE cells (Lot 090621) were thawed, washed with MDBK-MMmedia, and resuspended with BSS-Plus. Resuspended RPE cells werecentrifuged and resuspended again with 400 μL of BSS-Plus in a freshmicrocentrifuge tube. A viable cell count was done on the cellsuspension, and the concentration was adjusted to ±5% of the targetconcentration. The rigid injection cannula was attached to a 1 mL TBsyringe aseptically, and 200 μL of the cell suspension were drawn upinto the syringe via the cannula. The remaining 200 μL in the tube waslabeled “Non-Cannula”. The cell suspension in the syringe was dispensedinto a new microcentrifuge tube at a rate of 10-15 μL over 10 seconds.

A viable cell count was cell count was done to the “Cannula-Injected”sample. From both the “Non-Cannula” and “Cannula” samples 10,000cells/well were seeded into 96 well-plates the cells were cultured inRPE-GM. Another cell count was done 3-4 days post seeding to assess thelong term survival status. The cell counts for the cannula-injectedsample and non-cannula injected sample are provided in Table 10.

TABLE 10 RPE Survival for Non-Cannula and Cannula Suspension Non CannulaCannula 800 cell/μL Nominal Day 0 (viable cells/μL) 825 782 (94.8%)* Day3 (total cells/well) 218875 201887 (92.3%)* 1000 cells/μL Nominal Day 0(viable cells/μL) 960 1000 (104.2%)* Day 4 (total cells/well) 297050282750 (95.2%)* *% non-cannula value

The number of viable cell/μL after the cannula passage was comparable tothe non-injected RPE cells as shown in the Table 9. Also, the number ofviable cells 3-4 days post seeding did not differ significantly. Thedata presented herein demonstrates that the needle/syringe and cannulasystems that may be used for administration of human RPE cells candeliver a cell dose up to 1×10⁵ human RPE cells in a small volume (e.g.,about 2-3 μL) without damage/loss of cells (e.g., cellviability/activity, cell adhesion to the syringe). In conclusion,cannula/syringe passage does not substantially affect the viability orsurvivability of RPE cells. This is consistent with the preclinical datawhich shows that following subretinal injection in rats and mice, RPEcells are seen both microscopically and using immunostaining using humanspecific antigens.

Example 17 RPE Cells are not Tumorigenic

The methods of producing RPE cells described herein remove ES cells fromthe RPE cell preparation, thereby reducing the risk of teratomaformation. This was confirmed by assays to detect the presence of hES inthe RPE cells described herein. The human RPE cells described hereinwere tested for tumor formation and no such tumors were detected.

NIH-III nude mice considered suitable for study were weighed prior tocell implantation. A total of 27 animals were treated with hES cells, 30animals were treated with RPE cells, and 10 animals were left untreated.After all implantation procedures were completed, 56 male mice (weighing19.6 to 26.0 g at randomization) were assigned to the respective controland treatment groups identified in the following table using a simplerandomization procedure for each group.

TABLE 11 SUMMARY OF ADMINISTRATION Group Number Injection Ter- Num-Treat- of Male Cell Number Volume mination ber ment Animals (perinjection^(a)) (μL) (Week) 1 hES cells 23 1 × 10⁵ 3 4, 12, 40^(b) 2 RPEcells 24 1 × 10⁵ 3 4, 12, 40^(b) 3 None  9 NA NA 40 (Control) ^(a)Cellswere implanted into the subretinal space of the right eye. ^(b)Sixanimals per group were euthanized 4 weeks after cell implantation, 7animals were euthanized 12 weeks after cell implantation, and theremaining animals were euthanized 40 weeks after cell implantation.hES—Human Embryonic Stem RPE—Retinal Pigment Epithelial NA—Notavailable/applicable

At necropsy, the animals were euthanized and necropsied sequentially butalternating groups. The animals were evaluated at 4, 12 and 40 weeks(which is the approximate lifespan of the animal models). As only oneeye from each animal was treated, each animal acted as its own control.

The hES cell group observed significant tumor formation in 100% of theanimals, some as early as 4 weeks. In contrast, the RPE treated animalsdid not form tumors out to the lifespan of the animals. Thus the humanRPE cells preparations do not pose a risk of tumor formation followingtransplantation. Accordingly, the human RPE cell preparations areacceptable for use in transplantation (e.g., therapeutic applications).

Example 18 The RPE Cells are Stable and Integrated in Animal Modelsafter Transplantation

A fundamental limitation on the success and usefulness of cell-basedtherapies (e.g., transplantation) is the inability of the transplantedcells to survive, maintain their phenotype, integrate, and functionfollowing transplantation. To assess the stability and integration ofRPE cells, following injection into the eyes of 22 mice, the presenceand phenotypic stability of the transplanted human RPE cells wasconfirmed by immunofluorescense (to detect human molecular markers) andPCR (to detect human DNA). At 1 week, 1 month, 3 month, and 9 month timepoints the hRPEs were be identified apart from other cells by means oftheir physical characteristics (e.g., by their mRNA and proteinexpression and presence of human DNA in a mouse model.)

Co-Immunofluorescence

In mouse eyes injected with human RPE cells, the human RPE cells wereidentified by positive co-immunofluorescence to human mitochondrialantigen and bestrophin antigen and located within the mouse retinalpigmented epithelial cell layer, subjacent to the retina, within theposterior chamber or within the remaining scar at 9 monthspost-injection. Under light microscopy, the morphology of the positivestaining cells was characterized as typically linear arrangements ofcuboidal cells with round nuclei that were displaced eccentrically bysmall golden-brown intracytoplasmic pigment, and were consistent withretinal pigmented epithelial cells.

Cells staining positive for both human mitochondria and bestrophin wereidentified as linear to small round aggregates within the RPE layer, insubretinal locations, within scar, or as small aggregates within theposterior chamber vitreous space. Specifically, immunotluorescent cellsconsistent with RPE were identified within the mouse RPE layer andsubretinal space in 8 of 12 mice eyes examined in this study. In 2 of 4mice eyes, RPE cells were also identified within the posterior chamberand in 1 of 4 mice, RPE cells were identified in scar. RPE cells werenot observed in 3 of 12 eyes prepared for staining.

Under bright field light microscopy, in all cases the morphology of thepositive-staining human cells was characterized as organized lineararrangements of 4 to 10 cuboidal cells with round nuclei that weredisplaced eccentrically by small golden-brown intracytoplasmic pigment,consistent with retinal pigmented epithelial cells. When associated withthe mouse RPE, the human cells displayed typical polarity along abasement membrane with basally located nuclei and apically locatedpigmented granules. The human cells could be distinguished from mouseRPE as the human cells appeared slightly larger with fewer and smalleryellow-brown pigmented granules compared to the mouse RPE. There was noevidence of abnormal growth in the sections examined under theconditions of bright field microscopy.

None of the isotype or negative antibody controls showed any specificstaining. The untreated eye was consistently negative for anyfluorescence.

Detection of Human DNA

Although there is wide inter-animal variation within all the cohorts,human DNA was detected in all transplanted mice tested, including the 22mice assayed at the final (nine month post-transplantation) time-point.DNA was generally higher in mice that received the 100,000 cell dosecompared to mice that received 50,000 RPE cells. There is a relativelyconsistent level of DNA present throughout the observation period out tonine months with no consistent increase or decrease in DNA content.Additionally, histopathological assessments confirm that RPE cellssurvived in animal eyes out to nine months.

TABLE 12 Human DNA Detected in Mouse Eyes Transplanted with RPE Cells† 1week 1 month 3 months 9 months Ani- Ani- Ani- Ani- mal DNA mal DNA malDNA mal DNA Treatment ID (pg) ID (pg) ID (pg) ID (pg) 50,000 614 5056970 3040 916 151 712 3762 RPE cells 672 815 958 621 930 959 716 772 6832743 960 2116 908 3064 727 70 615 1647 952 4763 945 334 742 303 668 738957 3160 — — 917 1984 mean 2200 mean 2740 mean 1127 mean 1378 SD 1790 SD1359 SD 1337 SD 1523 100,000 611 1754 984 2135 920 3100 711 16025 RPEcells 609 3827 956 6161 918 880 717 4139 680 11104 977 3005 925 1184 73611278 608 15377 978 1250 910 5195 747 3290 622 12419 972 3809 929 6248921 805 mean 8896 mean 3272 mean 3321 mean 7107 SD 5831 SD 1877 SD 2280SD 6325 †The inter-animal variation within all the cohorts (e.g.,apparent different levels of DNA observed among the three groups) is notconsidered significant and is attributed to variability in the surgicalprocedure which may impact cell survival.

The eyes receiving the transplanted human RPE cells displayed healthyswathes of bestrophin positive cells with typical RPE morphology. Notumors were detected in this group or any other cohort except miceinjected with the 100% hES dose.

These data show that the NIH-III mouse model supports the survival ofthe injected human RPE cells for a significant time interval. A majorobstacle to developing a stem cell-based therapy for degenerativeretinal disorders is the poor integration and differentiation of retinalstem cells transplanted into recipient retinas. The RPE cells describedherein, in contrast, are well tolerated, stable, and integrate into thepatent after administration without tumor formation.

Example 19 Human RPE Cells Survive Long-Term Post-Injection in OcularTissues

A limitation on the success and usefulness of cell-based therapies(e.g., transplantation) is the inability of the transplanted cells tosurvive long-term following transplantation and the risk of teratomaformation. The purpose of this example was to identify, localize andcharacterize the morphology of RPE cells after 1, 3 and 9 monthspost-injection. The transplanted human RPE cells survived inrepresentative animals up to over 200 days, with no evidence of tumorformation or non-retinal human cells in the eyes. Cell proliferation wasevaluated at the 9 month time point for animals evaluated in theutilizing Ki67 staining. No proliferation was seen in either of thesestudies.

Selected ocular tissue sections were stained for the presence of humanmitochondria, human bestrophin, and human Ki67. Anti-human mitochondrialstaining was used as a clear marker for confirming human cell origin.Bestrophin is a basolateral plasma membrane protein expressed in retinalpigment epithelial cells, and was used to confirm RPE origin. Ki67 is awell recognized cell proliferation marker. See, e.g., Magdelénat (1992)J. Immunol. Methods 150(1-2):133-43.

Immunofluorescence staining was chosen over immunoperoxidase stainingfor demonstration of the antigens due to the presence of pigment in thecells of interest and to facilitate double staining of sections forbestrophin and Ki67. Ki67 staining in this study was only conducted atthe 1 and 3 month timepoints.

Positive and negative control tissues showed specific, sensitive andreproducible staining with minimal nonspecific background staining.Cells stained for human mitochondria as bright red punctate cytoplasmicstaining viewed with Cy3 580 nm filter. Cells stained for bestrophin asbright green basolateral membrane staining viewed with Zlexa488/Dylight488-550 nm filter. Ki67 staining was specific for nuclei andwas bright green under the same filter. Antibodies appeared to behuman-specific as there was no cross-reactivity with mouse tissue.However, some background staining was encountered in some sections,usually associated with retinal photoreceptors, vessel walls, collagenor skeletal muscle, but it was easily distinguished based on level ofbrightness, staining pattern and location.

None of the isotype or negative antibody controls showed any specificstaining. The untreated eye was consistently negative for anyfluorescence.

At all time points, cells staining positive for both human mitochondriaand bestrophin were identified as linear to small round aggregateswithin the RPE layer, in subretinal locations, occasionally withinscars, or as small aggregates within the posterior chamber vitreousspace. In all cases, the morphology of these human cells wascharacterized as organized cuboidal epithelial cells with round nucleidisplaced by small golden-brown intracytoplasmic pigment, consistentwith pigmented epithelial cells. When associated with the mouse RPE, thehuman cells displayed typical polarity along a basement membrane withbasally located nuclei and apically located pigmented granules. Thehuman cells could be distinguished from mouse RPE as the human cellsappeared slightly larger with fewer and smaller yellow-brown pigmentedgranules compared to the mouse RPE.

At the 1 month time point, the RPE cells were readily identified aslinearly organized cells within the RPE and/or subretinally in 5 of 6mice dosed with 100,000 cells and nuclear Ki67-positive staining wasobserved in 4 out of 5 mice eyes in which RPE cells were identified. Inmice dosed with 50,000 cells, RPE cells were observed in 3 of 6 miceeyes, and Ki67-positive cells were also observed in these same 3 miceeyes.

At the 3 month time point, most of the slides had moderate sectioningartifact but small aggregates of RPE cells were identified within theRPE and/or subretinal space in 2 out of 6 mice dosed with 100,000 cellsand 3 out of 6 mice dosed with 50,000 cells. Ki67 staining was performedfor 2 mice dosed with 100,000 cells and 4 mice dosed with 50,000 cells:Ki67 positive staining was observed in human RPE cells in 4 of 6 mice.No staining for Ki67 was observed in 2 mice in which staining for RPEwas adequate (both in the 50,000 cell dose group). In 2 mice dosed with100,000 cells, only few RPE cells were identified and consideredinadequate to assess Ki67 status.

At the 9 month time point, immunopositive RPE cells were identifiedwithin the mouse RPE and/or subretinal space in 5 of 6 mice eyes dosedat 100,000 cells and 2 of 5 mice eyes dosed with 50,000 cells. In 1 of11 mice (animal number 743 dosed with 100,000 cells) immunopositive RPEcells were identified in the posterior chamber and scar; and in 1 animal(animal number 744 dosed with 50,000 cell) RPE cells were only observedin the scar. RPE cells were not identified in 2 of 5 eyes prepared forstaining in 50,000 cell group at the 9 month time-point. Ki67 stainingwas not performed for this group of slides.

Conclusion

In mouse eyes injected with human retinal pigmented epithelial cells,RPE cells were identified by positive co-immunofluorescence to humanmitochondrial antigen and bestrophin antigen and located within themouse retinal pigmented epithelial cell layer, subjacent to the retina,within the posterior chamber or within the remaining scar up to 9 monthspost-injection. Under bright field light microscopy, the morphology ofthe positive staining cells was characterized as typically lineararrangements of cuboidal cells with round nuclei that were displacedeccentrically by small golden-brown intracytoplasmic pigment, and wereconsistent with retinal pigmented epithelial cells. A subset of thesecells showed nuclear positivity for the proliferation marker Ki67 at 1and 3 months after injection.

Function was deteriorating down towards baseline levels by 180 days ofage (i.e., 160 days post-transplantation). At the point where functionwas diminished, there were no signs of pathological manifestations.

The appearance of the remaining retina was also examined. There were nountoward manifestations. Photoreceptor survival was evident in most ofthe transplanted animals although donor cell survival as seen by humannuclear marker staining was less frequent. There was no indication ofextraneous cell growth or of abnormal cell patterns within the innerretina.

Normal retinal appearance was observed in RCS rats (i.e., no vascularabnormalities, laminar disorder) in the area where the transplantedcells were introduced, in spite of the fact that donor cells were nolonger evident in some of these eyes. Photoreceptors, although present,were fewer in number than would typically be seen at 100 days of ageafter P21 transplants. There was no evidence in any of the eyes examinedof potentially tumorous growth of the donor cells.

Human pigmented epithelial cells were identified within segments of ratretinal pigmented epithelial cells, and thus confirm the presence ofhuman cells in representative animals up to >220 days post surgery. Thecells were consistent with RPE morphology and positive for bestrophin.Therefore, the human RPE cells described herein may be transplantedwhere they integrate forming stable, functional retinal pigmentedepithelial layer.

TABLE 13 Long-Term Survival of hRPE cells in mouse eyes Animals withAnimals with human human Number cells found cells surviving of in theeye in the eye Survival Time Animals (number) (%) 1 month (4 weeks) 2626 100% 2 months (8 weeks) 19 19 100% 3 months (12 weeks) 28 28 100% 9months (40 weeks) 52 48  92%

Unlike other transplant locations, the eye is a small organ and thenumber of cells that may be implanted into the subretinal space is quitesmall (e.g., 100,000 RPE cells) compared to millions of cells that maybe injected into other sites for other conditions. Additionally, thesurvival rate of transplanted cells (e.g., xenogenic, allogeneic,syngeneic, or autologous) in various animal models is generally low.Although donor cells may be easily detected immediately aftertransplantation (e.g., several days out to 3 weeks), there is aprogressive loss of survival over time, generally resulting in less than1% long-term survival in animal model studies. For example, Wang, et at(2005) Invest Opthalmol Vis Sci 46(7): 2552-60 reported a loss ofsurviving human RPE cells in immunosuppressed RCS rat eyes from 5% at 6weeks post transplantation to 0.2% at 28 weeks. Carr, et al. (2009) PLoSOne 4(12): 8152 disclosed that human iPS-RPE cells were undetectable 13weeks post-transplantation. Del Priore, et al. (2003) Invest OpthalmolVis Sci 44(9): 4044-53 found <1% of porcine RPE cells in rabbit eyemodel after 12 weeks and Canola, et al. (2007) Invest Opthalmol Vis Sci48(1): 446-54 showed only 0.44% of injected cells survived at 3 months.In the methods described herein, only a portion of the transplanted RPEcells (e.g., >1%) may survive long-term (e.g., over 9 months). Theinventors surprisingly discovered, however, that only a small number ofcells are required to affect visual improvement.

Example 20 Evaluation of Various Delivery Procedures

The purpose of the example was to examine the subretinal injection ofRPE cells in non-human primates, in particular vitrectomy, a method tocreate a subretinal bleb, and cell doses. The risk of stem cell graftrejection and the presence of any deleterious effects on the retinalphysiology as a consequence of cell injection was also examined. Thestudy used 8 animals (16 eyes). All animals were injected according tothe following schedule in Table 14.

TABLE 14 Animal Injection Schedule Right Eye Left Eye Number of BlebsNumber of blebs (50,000 cells) and (50,000 cells) Age position relativeto and position relative Cyclo- Animal # (Yrs) macula/optic disc tomacula/optic disc sporine A  2 1 submacular 2 superior None B  2 1submacular 2 superior, inferior. None 1 nasal C 17 1 superotemporal 1superotemporal None D 16 1 superior, 1 superior, None 1 temporal, 1temporal 1 nasal to optic disc E 13 2 superior 2 superior 5 mg/kg IM SIDF 16 1 superotemporal. 1 inferior, 5 mg/kg 2 nasal to optic disc 1inferonasal to IM SID optic disc G 16 2 superior, 2 superior, 5 mg/kg 1inferior 1 inferonasal IM SID H 15 2 inferior, 2 inferior, 5 mg/kg 1nasal to optic disc 1 nasal to disc IM SID

The surgeries were done on two days: on the first surgery day thefollowing steps were followed: After the animal was intubated, the areaaround the eyes were prepped with iodine solution. A 1060 drape was usedto drape the animal for ophthalmic surgery. For each of the animal, theright eye was done first then the left. A Barraquer-type speculum wasinserted. A peritomy was created in the superotemporal quadrant. Thescleral bed was cauterized with wet-field cautery to achieve hemostasis.A sclerotomy was created 3 mm posterior to the limbus with a 20 gaugeMVR blade. A plug was placed and a similar procedure was done in thesuperonasal quadrant to create a peritomy and a sclerotomy.

For some subjects, vitrectomy was performed using an end-irrigatinglight pipe, a vitrector, and a hand-held irrigating contact lens in aneffort to elevate the posterior hyaloid. Then a 19 gauge end-irrigatinglight pipe, a Synergetics subretinal injector, and a Machemer irrigatingcontact lens were used to create subretinal blebs. Then a subretinalpick was used to inject the cells. Then the sclerotomies were closedusing 6-0 Vicryl sutures and the conjunctival peritomy with a 6-0 plaingut sutures. Zinacef (Cefuroxime, 125 mg) and Decadron (Dexamethasone,10 mg) were given as subconjunctival injections OU. Erythromycinointment was placed over the eyes OU.

On the second day of surgery, the following steps were followed for eachprocedure: After the animal was intubated, the area around the eyes wasprepped with iodine solution. A 1060 drape was used to drape the animalfor ophthalmic surgery. For each of the animal, the right eye was donefirst then the left. A Barraquer-type speculum was inserted. A peritomywas created in the superotemporal quadrant. The scleral bed wascauterized with wet-field cautery to achieve hemostasis. A sclerotomywas created 3 mm posterior to the limbus with a 20 gauge MVR blade. Aplug was placed and a similar procedure was done in the superonasalquadrant to create a peritomy and a sclerotomy. Then a 19 gaugeend-irrigating light pipe, a Synergetics subretinal injector, and aMachemer irrigating contact lens were used to create subretinal blebs.Then a subretinal pick was used to inject 50 micoliters of stem cells(2000 cells/microliter) into each of the blebs. Then the sclerotomieswere closed using 6-0 Vicryl sutures and the conjunctival peritomy witha 6-0 plain gut sutures. Zinacef (Cefuroxime, 125 mg) and Decadron(Dexamethasone, 10 mg) were given as subconjunctival injections OU.Erythromycin ointment was placed over the eyes OU. Following eachsurgery retinal photos and ERGs were done. At termination all animalsunderwent full necropsy and the eyes were examined histologically.

To summarize, the technique was refined to be a two port pars planaapproach with an irrigating light pipe and subretinal cannula, and wehave histologically confirmed successful implantation to the subretinalspace. A vitrectomy may also be performed, if desired.

One suitable method for subretinal bleb formation was as follows: theretina may be approached with the Synergetics subretinal cannulaconnected to a Hamilton 1 ml syringe with a screw plunger containingBalanced Salt solution (BBS). The BSS may be injected slowly creating aretinotomy and then a small subretinal bleb is raised. This may minimizeretinal trauma. The cannula may be then introduced through theretinotomy and the BSS injection restarted and continued to expand thebleb to the correct volume. A process of gentle retinal massage releasesthe tension in the bleb. The Synergetics cannula may be removed and a30-gauge Hurricane Instruments needle connected to tubing and syringepreloaded with cells may be introduced. The cells may be infused overabout one minute under direct viewing to ensure correct cannulapositioning and minimize reflux. This instrumentation procedure issuitable for use in humans.

Retinal photography and electrophysiology were performed on each eyepreoperatively and at the 2-week and one-month time points. Completeretinal reattachment was noted within 24 hours and multifocal ERGrecordings show no electrophysiological evidence of pathology. In total,fifteen eyes of eight adult rhesus macaques underwent histologicalexamination; one eye developed endophthalmitis and was excluded from thestudy. BrdU labeling was used to detect the human RPE cells. Cells wereobserved localized to the subretinal space and are associated withretinal reattachment, excellent preservation of retinal morphology, andlack of inflammation or rejection.

Example 21 RPE Cells in Photoreceptor Rescue in the RCS Rat Model

At postnatal day 21-23 (P21 —23), RCS rats (n=14) were anesthetized andreceived subretinal injections of 20,000 hRPE cells/eye via atrans-scleral approach into the upper temporal retina area. Control ratsreceived an injection of medium alone (n=8). Non-dystrophic congenicrats were available for comparison. All animals received dailydexamethasone injections (1.6 mg/kg, i.p.) for 2 weeks and weremaintained on cyclosporin-A administered in the drinking water (210mg/L; resulting blood concentration: 250-300 μg/L) days prior to cellinjection until animals were euthanized.

To test visual function, the electrical activity of the outer (a-wave)and inner (b-wave) retina in response to light flashes was tested by ERGresponses at both P60 and P90. At P60, the a-wave ERG response isnormally lost in RCS rats, and by P90, the b-wave response is severelydepleted, allowing graft-related effects to be recognized overbackground performance. By P60, hES-RPE grafted animals achievedsignificantly better responses over sham-injected animals (p≤0.05,t-test) for a-wave (31±27 vs. 6±17 V), b-wave (108±46 V vs. 36±33 V) andcone b-wave (57±19 vs. 28±13 V) (FIG. 9).

The optomotor test was used to provide a measure of spatial acuity. OnP100 sham-injected rats, a threshold response of 0.29±0.03 c/d wasrecorded and untreated animals gave a figure of 0.21±0.03 c/d. Bycontrast, the cell-grafted rats sustained levels of 0.42±0.03 c/d,significantly better than sham injected rats (p≤0.05, t-test) (FIG. 10).

Average and best performers in the optomotor test were selected fromeach group for luminance threshold response testing. Results wereobtained from animals receiving hRPE cells (n=7), sham injections (n=5),and no treatment (n=6). In non-dystrophic rats, a threshold response ofless than 0.6 log units is recorded. On P100, untreated RCS rat neuronsacross the whole visual field failed to respond with thresholds of 2.7log units or better, while responses could be elicited from 18% of thearea in sham-injected rats. By comparison, the cell-injected rats showed52% of the collicular area with thresholds of 2.7 log units or better,with a best point of 1.3 log units (FIG. 11).

Histological examination of the retinas demonstrated the presence ofhuman specific nuclear marker that also stained for RPE-specific markers(RPE65 and bestrophin). Staining with human-specific proliferating cellnuclear antigen (PCNA) was negative, indicating that there was noproliferation of the hRPE cells. In addition, the histology revealedpersistence of the cell population without inflammation or immune cellinfiltration and without cellular proliferation or tumor formation.

The results of this study indicate that there was significant visualrescue above controls as determined by all three functional assessments.The cells survived long-term (>100 days) after transplantation into RCSrats, and localized to the subretinal space without migration into theretina. In addition to extensive photoreceptor rescue (5-7 cells deep inthe outer nuclear layer), the relative acuity as measured by theoptomotor system showed that animals treated with hES-derived hRPEperformed significantly better than sham and untreated controls (50% and100% improvement in visual performance, respectively; visual acuity wasapproximately 70% that of normal non-dystrophic rats). There was also noevidence of any tumor formation.

In these experiments, the transplantation of RPE cells resulted in themaintenance or improvement of visual function. Therefore RPE cellsdescribed herein may be used in a cell therapy for treating retinaldegenerative disease such as the amelioration of age-related maculardegeneration (AMD) and senile macular degeneration (SMD).

Example 22 Long-Term Safety and Function of RPE from Human EmbryonicStem Cells in Preclinical Models of Macular Degeneration

Summary of Results

The RPE cells described herein may be used for the treatment ofage-related macular degeneration and Stargardt's disease. Here we showlong-term functional rescue using hESC-derived RPE in both the RCS ratand Elovl4 mouse, animal models of retinal degeneration and Stargardt's,respectively. Good Manufacturing Practice-compliant hESC-RPE survivedsubretinal transplantation in RCS rats for prolonged periods (>220days). The cells sustained visual function and photoreceptor integrityin a dose-dependent fashion without teratoma formation or untowardpathological reactions. Near-normal functional measurements wererecorded at >60 days survival in RCS rats. To further address safetyconcerns, a Good Laboratory Practice-compliant study was carried out inthe NIH IR immune-deficient mouse model. Long-term data (spanning thelife of the animals) showed no gross or microscopic evidence ofteratoma/tumor formation after subretinal hESC-RPE transplantation. SeeLu, et al. (2009) Stem Cells 27: 2126-2135.

Animals and Experimental Designs

Pigmented dystrophic RCS rats (n=79) and ELOVL4 mice (n=28) were used inthe main experiments. NIH III immunonude mice (n=45) were used forsafety study. For RCS rats, animals were divided into live groupsaccording to the doses they received. They were 5×10³ (5,000)/eye(n=21), 2×10⁴ (20,000)/eye (n=21), and 5×10⁴ (50,000)/eye (n=21).Animals from all dosage groups received cells with low medium and highpigmentation. All above the dosage group animals were received cellswith low, medium and high pigmentation (Table 14). For furthercomparison, two groups were added: one group of animals (n=8) received7.5×10⁴ (75,000)/eye cells and another group (n=8) received 1×10⁵(100,000)/eye cells with medium pigmentation. For ELOVL4 mice, the eyesreceived 5×10⁴ (50,000)/eye cells with medium pigmentation. All animalsin the main experiments were maintained on oral cyclosporine Aadministered in the drinking water (210 mg/l, resulting bloodconcentration of ˜300 μg/l) from 1 day before transplantation until theywere sacrificed. An intraperitoneal injection of dexamethasone was givenfor 2 weeks (1.6 mg/kg/day) after surgery in cell and control injectedrats and for 2 weeks alone in untreated animals. All animals weremaintained under a 12-hour light/dark cycle.

Cell Preparation

Culture of hES cells and Differentiation into Mature RPE Cells.

All cell manufacturing procedures were carried out in ISO Class 5biosafety cabinets in an ISO Class 7 clean room facility under strictenvironmental control monitoring systems and a routine microbial testingregimen. Single-blastomere hESC lines MA01 and MA09 were maintained aspreviously described herein. hES cells were dissociated from the primarymouse embryonic fibroblast layer by treatment with 0.05% trypsin-EDTAand were seeded in 6-well low-attachment plates to allow EB formation ina chemically defined minimal essential medium (MEM)-based medium(MDBK-GM) containing B-27 supplement for about 7 days and plated ongelatin-coated (0.1%) dishes until RPE colonies were visible. RPE waspurified by 3-hour exposure to 4 mg/ml type IV collagenase and manuallyisolated with a glass pipette. Purified RPE was seeded ontogelatin-coated tissue culture plates and expanded in EGM-2 medium untildesired density was achieved, at which point cultures were reverted toMEM-based medium (MDBK-MM) and cultured until the appropriate phenotypewas achieved. RPE was dissociated from culture using a 1:1 mixture of0.25% trypsin-EDTA and Hanks-based cell dissociation buffer and wascryopreserved in 90% fetal bovine serum and 10% dimethylsulfoxide

Quantitative, Real-Time, Reverse Transcription-Polymerase ChainReaction.

RNA was extracted from the cells using TRIzol reagent according to themanufacturer's protocol. Eluted RNA was quantitated byspectrophotometry, and 10 μg was subjected to DNase digestion, followedby a reverse transcription reaction using a QUANTITECH® reversetranscription kit with a mixture of oligodT and random hexamers primers.Fifty NANOGrams per well of cDNA was used as templates in quantitativepolymerase chain reactions (qPCRs) with oligonucleotides specific forhESC and retinal genes. All qPCR reactions were performed in triplicate,with the resultant values being combined into an average threshold cycle(CT). The efficiency of qPCR was calculated from the slope of a relativestandard curve using GAPDH primers. Relative quantization was determinedusing a STRATAGENE® MX3005P QPCR system measuring real-time SYBR Greenfluorescence and calculated by the ΔΔCT method. Fold differences arecalculated using the ΔΔCT in the formula 2−ΔΔCt. Expression profiles forthe mRNA transcripts are shown as fold differences in comparison to mRNAlevels in hES cells.

Microarray Gene Expression Profiling.

Global gene expression analysis was performed using the humanAFFYMETRIX® HGU133 Plus 2.0 microarray platform on both of the singleblastomere-derived hESC lines MA01 and MA09 and the resulting RPE cellsderived from each. Additionally, fetal RPE, ARPE-19, and retinoblastomacell lines were used as controls

Western Blot Analysis.

Immunoblot analysis was carried out using standard SDS-PAGE methodsusing the BIO-RAD® Mini-Protean and Mini-Transblot Cell. The proteinbands were visualized using Western Lightning Chemiluminescence Reagentand a KODAK® 4000 MM digital imaging station. Commercially availableantibodies specific for DPPA4, TDGFI β-actin, CHX-10, Otx2, REX1, RPE65,PAX6 Bestrophin, CRALBP, Pax2, MitF, NANOG, Oct4, PEDF, and Tyr as wellas horseradish peroxidase-conjugated secondary antibodies were used.

TABLE 15 Number of eyes treated at each pigment level Number of cellsLow pigment Medium pigment^(a) High pigment  5,000 8 6 7 20,000 7 8 650,000 7 7 7 Sham 12  12 11 Untreated 9 10 10 ^(a)Sixteen additionaleyes in the “”medium pigment” group were also treated with a higherdosage: 75,000 (n = 8) and 100,000 (n = 8) cells.Transplantation Protocol

Before cell transplantation, cells were thawed and washed in balancesalt solution (BSS) and suspended in BSS. Three cell lines designatedlow, medium, and high pigment were given in different dose groups. Theseare summarized in Table 15. Using techniques known in the art, asuspension of cells was delivered into the subretinal space of one eyethrough a small scleral incision, suspended in 2 μl of BSS medium usinga fine glass pipette (internal diameter, 75-150 μm) attached by tubingto a 25-μl Hamilton syringe. The cornea was punctured to reduceintraocular pressure and to limit the efflux of cells. A sham-surgerygroup was treated the same way, except the carrying medium alone wasinjected. Pigmented dystrophic RCS rats received unilateral subretinalinjections of the cell lines (n=79 eyes) at P21; control rats receivedsham alone (n=35 eyes) or were untreated (n=29 eyes). Elovl4 mice at P28received cells (n=12 eyes), sham alone (n=8 eyes), or were untreated(n=8 eyes). Immediately after injection, the fundus was examined forretinal damage or signs of vascular distress. Any animal showing suchproblems was removed from the study and excluded from the final animalcounts.

Spatial Visual Acuity.

Animals were tested for spatial visual acuity using an optometry testingapparatus comprising four computer monitors arranged in a square, whichprojected a virtual three-dimensional space of a rotating cylinder linedwith a vertical sine wave grating. Unrestrained animals were placed on aplatform in the center of the square, where they tracked the gratingwith reflexive head movements. The spatial frequency of the grating wasclamped at the viewing position by recentering the “cylinder” on theanimal's head. The acuity threshold was quantified by increasing thespatial frequency of the grating using a psychophysics staircaseprogression until the following response was lost, thus defining theacuity. Rats were tested from P60 to P240 at monthly intervals. Elovl4mice were also tested in this apparatus at 3, 5, 7, and 11 weeks aftersurgery.

Luminance Threshold.

This was studied to provide a different measure of function from thespatial acuity and was achieved by recording single and multiunitactivity close to the surface of the superior colliculus (SC) usingglass-coated tungsten electrodes (resistance: 0.5 MΩ; bandpass 500 Hz to5 KHz) with previously described procedures. Recordings were made onlyin rats, selected on the basis of good and representative optomotorresults: mice were not examined with this test. The brightness of a 5°spot was varied using neutral density filters (minimum steps of 0.1 logunit) over a baseline level of 5.2 log units until a response double thebackground activity was obtained: this was defined as the thresholdlevel for that point on the visual field. A total of 15-20 positionswere recorded from each SC. All animals were recorded at about P100, andsome were studied again at a second time point at about P190. Data areexpressed as a graph of percentage of SC area with a luminance thresholdbelow defined levels and as raw results.

Histology.

At the end of functional tests, all animals were euthanized with anoverdose of sodium pentobarbital and perfused with phosphate-bufferedsaline. The eyes were removed, immersed in 2% paraformaldehyde for 1hour, infiltrated with sucrose, embedded in optical cutting temperature,and cut into 10-μm horizontal sections on a cryostat. Four sections (50μm apart) were collected per slide, providing five series of everyfourth section collected. One was stained with cresyl violet forassessing the injection site and integrity of retinal lamination. Theremaining slides were used for antibody staining, following previousprotocols, and were examined by regular and confocal microscopy.

Safety Study.

Cells were prepared and transplanted using the same methodologydescribed above for the RCS rat study. A minimum of six NIH III mice pergroup were injected with either hES cells or hESC-derived RPE from theMA09 single blastomere cell line in three time-based cohort groups(n=36). The animals were killed by CO₂ inhalation followed byexsanguination at 1, 3, and 9+ months based on cohort. Three negativecontrol animals were also put in the study for each cohort (n=9). Lifestudy assessments included routine clinical assessments and body weightanalysis, plus pre-sacrifice clinical chemistry. Post mortem, eyes wereremoved and immersed in cold 4% paraformaldehyde, for up to 1 week. Thetissue was embedded in paraffin and sectioned. Select slides werestained with hematoxylin and eosin. Slides were examined microscopicallyto assess retinal lamination and tumor formation.

Differentiation and Characterization of hESC-Derived RPE

Human RPE cells were generated using a cGMP-compliant cellularmanufacturing process. Three different batches of RPE were created fromeach blastomere-derived hESC line based on morphological assessment ofpigmentation (FIG. 15), an important indicator of RPE maturation.

Each production run generated about 50×10⁶ RPE cells from a singlefrozen ampule of 1×10⁶ hES cells. This amount is sufficient to doseabout 500 rats or 50-100 human subjects. Additionally, the methodsdescribed herein are completely suitable to available scale-uptechnologies such as bioreactor culture or large-scale fluid handlingsystems.

To characterize the developmental stages during RPE differentiation,several assays were used to identify the expression levels of genes keyto each stage of development. qPCR was developed to provide aquantitative and relative measurement of the abundance of celltype-specific mRNA transcripts associated with the RPE differentiationprocess. A panel of genes associated with hESC pluripotency (Oct-4,NANOG, Rex-1, TDGF1, Sox2, DPPA2, and DPPA4), neuroectodermintermediates (PAX6 and Chx10), and RPE (RPE-65, Bestrophin, CRALBP,PEDF, MitF, Otx-2, Tyr, and Pax2) was established and assayed for eachby qPCR. With regard to quality control of cellular manufacturing, themarked decrease in all stem-related genes and concomitant increase inall retinal-associated genes, at a level of 10- to 100-fold, was deemedacceptable release criteria.

FIG. 13 shows the gene expression profile of the transcripts duringdifferentiation to mature RPE, including samples from hES cells (d0),embryoid bodies (EBs, d7), plated EBs (d14), mixed population of newlyformed RPE and less differentiated cells (mixed, d28), purified earlyRPE (eRPE, d35), and fully matured pigmented RPE (mRPE, d56). Aprogressive decrease in the expression level of hESC-specific genes(FIG. 13A) was accompanied by an increase in the level of neu-roectodermand RPE-specific genes. Lightly pigmented RPE (FIG. 12) expressed1,000-fold lower quantities of Oct-4, NANOG, Sox2, and DPPA4;<10,000-fold less TDGF1; and 50-fold less Rex-1 and DPPA2 than hESC. Thecells also expressed 10- to 100-fold greater quantities of RPE65,CRALBP, PEDF, Bestrophin, PAX6, and MitF and expressed >100,000,000-foldTyr, a downstream target of MitF/Otx2 in RPE. This cell populationexpresses genes such as PAX6 and CHX10 because this stage represents an“immature” population of RPE derived from embryonic cells, and maycontinue to express markers associated with developing cells of theneuretina and/or neurectoderm.

The phenotypic changes that RPE undergoes during the in vitro maturationprocess were characterized by qPCR (FIG. 12A-C). FIG. 12A shows that RPEwith a higher degree of pigmentation and polygonal cell borders(corresponding to FIG. 12C) maintains higher expression of RPE-specificgenes. Notably, both pigmentation and the high level of RPE-specificgene expression are correlated with the emergence of Pax2 expression anda sharp increase in MitF, Otx2, and Tyr expression. MitF expression, andin turn Tyr, is achieved in RPE through synergy of Pax2 and PAX6 duringembryonic development.

Proteomic Validation of Selected Transcripts in hESC-Derived RPE

To verify that genes of interest were expressed at the protein level,all targets of the initial transcriptional profile panel were assayed byWestern analysis. As an internal control, hESC-derived RPE was comparedwith the ARPE-19 cell line by both qPCR and Western analysis. FIG. 10Ashows that, although hESC-RPE expresses similar levels of RPE-specifictranscripts to ARPE-19, the hESC-RPE expresses more abundant levels ofthese proteins (RPE65, PEDF, Pax2, and Bestrophin). Additionally,proteins expressed by hES cells are all downregulated in the finaldifferentiated cell product. This disappearance of stem-related proteins(by immunoblot) and concomitant emergence of retinal-associated proteinsis indicative of RPE cells as described herein.

Bioinformatic Analysis of Global Gene Expression in hESC-RPE

The biological relevance of the morphological changes observed in vitrowere assessed by gene expression profiling and subsequent informaticanalysis of both hESC lines, each with three different morphologies: acontrol and several “reference” cell lines on the human Affymetrix®HG-U133 Plus 2.0 microarray platform. FIG. 14 shows a principalcomponent analysis (PCA) scatter plot, indicating the contribution tovariance that the two major variables, cell type and cell line (x- andy-axis, respectively), yield on global gene expression. A linearprogression was observed from the undifferentiated (hES cells) statethrough the three levels of RPE pigmentation. Interestingly, thedepigmented RPE cells (See FIG. 12A) cluster closer to both ARPE-19 andfetal RPE; the latter display similar Morphological characteristics tothis batch of cells in vitro. The more heavily pigmented batches of RPEcells appear to cluster farther from hES cells and retinoblastoma cells(RB) than any other cell type tested. Whereas the pigmented batches ofRPE from MA01 and MA09 do not overlap by PCA, they are within a similarorder of magnitude to each other to that of fetal RPE and ARPE-19. Takentogether, these data suggest that the more heavily pigmented hESC-RPEcells may be considered the most differentiated, and from a safetystandpoint, the most genetically divergent from cells possessing“sternness” or expressing cancer-related genes.

Pathogen Testing and Stability of RPE

An important criterion to consider in the use of RPE cell preparationsfor therapeutic applications is product safety (e.g., contamination orinfection with viral or bacterial agents). To ensure that the RPE cellswere free of contamination during the extensive culture anddifferentiation process, the following testing according to U.S. Foodand Drug Administration and International Conference on Harmonizationguidelines for applicable microbial and viral agents were conducted:United States Pharmacopeia membrane filtration sterility,fluorochrome-based mycoplasma, transmission electron microscopy forviral particles, in vitro tissue culture safety testing for adventitiousagents, in vivo inapparent virus detection, PCR-based reversetranscriptase detection, HIV-1, HIV-2, HBV, HCV, CMV, HTLV-1 and -2,parvovirus B19, Epstein-Barr virus, and herpesvirus 6. Additionally, thecells were cytogenetically analyzed by G-banding karyo-type analysis.Results confirmed that these cell lines are karyotypically stable andsubstantially free of infectious pathogens.

Dosing Studies in RCS Rats.

The effect of different doses on efficacy was titrated using theoptomotor response as an indicator. The results at P90 (70 days aftertransplantation) are summarized in FIG. 15A. Improved rescue of spatialacuity occurred from 5,000 to 50,000, after which even doubling the doseof cells to 100,000 had no significant effect on efficacy. Performersamong the cell-injected group gave a figure of 0.536 cycles/degree (c/d)compared with 0.6 c/d in normal rats, which is about 90% of normalvalue. There was no significant difference between sham and untreatedgroups, which performed significantly worse than the cell-injected group(p<0.01).

Luminance thresholds were also measured in a subset of rats selected bytheir performance on the optomotor response. An area with highsensitivity corresponded to the area of retina in which the cells wereintroduced, as indicated in FIG. 15C-15F. For statistical comparison thedata for this part of the example is presented as a percentage of thearea of the visual field representation from which thresholds betterthan designated levels were recorded without regard to position. Thisgives a simple indicator of overall efficacy, as well as a responsefigure, dissociated from spatial considerations. It is clear that theoverall sensitivity recorded at 50,000 is superior to 20,000, but aswith spatial acuity, it does not change significantly between 50,000 and75,000. For example, about 45% of the SC gave thresholds of 2.2 logunits with 50,000 cells/eye and about 40% with 75,000 cells/eye.Generally, the mean response levels at 100,000 were better and gave morelong-lasting rescue than did lower doses. See FIGS. 15 and 16.

Pigmentation Results

There was no significant difference between pigment groups on visualacuity (FIG. 17), however, compared to the sham or untreated controls,all pigment groups did show significantly better visual acuity at alltime points between P40 and P240.

Batch and Longevity of Effect in RCS Rats

Although slight differences in optomotor acuity were seen between thedifferent pigment levels (Table 14), they were not significant. Incontrast, there was a significant difference at all time points studiedbetween the cell-injected groups and medium-injected and untreatedcontrols. See FIG. 16. Over time, there was a reduction in acuityresponse for all the cell groups and dose levels.

To examine how luminance responses deteriorated with time, thresholdswere recorded at two time points in individual rats. An example is shownin FIG. 18. As shown, the luminance thresholds show seriousdeterioration on the untreated side, with more than one half the areabeing nonresponsive at P187 compared with P98, whereas responsiveness isstill sensitive on the cell-injected side, although some reduction inthresholds has occurred (0.7 log units at P98 vs. 1.0 log units atP187). Raw data from an animal that received cell injection: luminancethreshold responses were recorded at P98 (shown in FIG. 18A) and P187(shown in FIG. 18B) in the same rat from multiple points within thesuperior colliculus (SC). This method quantifies functional sensitivityto light across the visual field of the eye. The topographical mapdepicts the luminance threshold responses (measured in log unitsrelative to background illumination of 0.02 cd/m²) at 15 and 16 pointsin the left and right sides, respectively, within the SC. In FIG. 18A,all points of luminance threshold responses in the treated side are lessthan 2.0 log units, whereas in the untreated side, all points aregreater than 2.3 log units. Table 15B depicts the same animal wasrecorded at P187 (>5 months after surgery); there is deterioration insensitivity to light compared to P98; however, it is still significantlybetter than the untreated fellow eye (which has no response over halfthe area). Abbreviation: c/d, cycles/degree. See FIG. 18.

Efficacy in Elovl4 Mice

Visual acuity in normal mice tested by the same optomotor device waslower than that in rats (0.35 vs. 0.6 c/d). In untreated Elovl4 mice,the visual acuity deteriorated as photo-receptor degeneration progressedfrom 0.34 c/d at P28 to 0.24 c/d at P105. FIG. 15B. Subretinal injectionof hESC-RPE improved the visual acuity over controls at all time pointstested. Cell-injected eyes had a figure of 0.32±0.04 c/d at P63 (5 weeksafter surgery) compared with 0.26±0.03 c/d in sham-injected anduntreated controls. FIG. 15B. Statistical analysis indicated that thedifference between cell-injected and controls was significant (t test,p<0.05).

Histological Examination of RCS Rats

General Retina Structure.

Retinal sections from cell-injected, sham, untreated, and normal controlrats were stained with cresyl violet and examined under lightmicroscopy. At P90, compared with normal control (FIG. 19A), thecell-injected retina had five to six layers of photoreceptors (FIG.19B), whereas the untreated retina had only a single layer remaining(FIG. 19C). In accordance with the functional results, the 5,000/eyedoses had slightly better photoreceptor rescue (FIG. 19D) thansham-operated (primarily with localized photoreceptor rescue aroundinjection site), whereas the 20,000/eye produced better photoreceptorrescue. The 50,000/eye and greater doses gave consistent photoreceptorrescue, covering a larger area of the retina (FIG. 19E, 19F) withpreserved cones. At P150, cell-injected retinas still had an outernuclear layer two to three cells deep, and the inner retina laminationwas not disrupted. In contrast, both untreated and sham-operated retinasshowed a typical secondary pathology, including abnormal vascularformation, RPE cells, and inner retinal neurons migrating along abnormalvessels, leading to distortion of retina lamination. At P240,cell-injected retinas still had an outer nuclear layer of one to twocells deep, and the inner retina still showed an orderly lamination. Incontrast, advanced degeneration was evident in control retinas: theinner nuclear layer became irregular in thickness, ranging from onelayer to multiple layers; RPE cells had migrated into the inner retina;and abnormal blood vessels were seen (FIG. 19I).

Antibody Staining.

The human specific nuclear marker, MAB1281 was used to identify thedonor cells. They formed a layer, one to two cells deep, and integratedinto the host RPE layer (FIG. 19G, 19H), as was seen in our previousstudy. Photoreceptor rescue continued beyond the limits of distributionof donor cells, suggesting that rescue was at least in part caused by adiffusible effect. Cone arrestin antibody showed that conephotoreceptors were preserved with disorganized segments (FIG. 19F) atP90. Donor cells were still evident up to at least P249 (FIGS. 19G and19H). There was no indication of continued donor cell division (e.g.,shown by the proliferating cell nuclear antigen marker).

Safety Assessment

Studies in NIH III Mice.

The long-term risk of teratoma formation was tested in the NIH III mousemodel. The NIH III mouse was chosen for its immune-deficient status; thenude mouse has three mutations rendering it devoid of T cells, NK cells,and mature T-independent B lymphocytes. However, the NIH III mouseretains eye pigmentation, which provides better visualization forsubretinal transplantation surgery. The surgical technique was the sameas performed in the RCS study. The study compared the hESC-RPE toundifferentiated hES cells (positive control) to determine the teratomaformation potential of the 100,000 RPE cell dose over three time points:1, 3, and 9 months (the approximate lifespan of the animal; n=6 percohort). In contrast to the animals that received undifferentiated hEScells, no teratoma or tumor formation was found in any of the animalsinjected with the hESC-derived RPE. In addition, basic animal safetyassessments were normal compared with controls.

Absence of Tumorigenic Growth in RCS Rats.

In the RCS rat transplant study, none of the 79 cell-injected retinasexamined, including the longest time points, showed any evidence ofuncontrolled cell proliferation. There was no evidence of teratomaand/or tumor formation.

Discussion

These results show the long-term safety and efficacy of hESC-derived RPEcells produced under manufacturing conditions applicable for use inhuman clinical trials are described herein. In addition to thedevelopment of assays with qualified range limits (which constituted the“identity” of the final RPE product), extensive pathogen testing wascarried out to ensure that the manufacturing procedure did not introduceany infectious diseases or adventitious agents into the RPE cells.

To confirm the functionality of these GMP-compliant cells, bothdose-response and long-term efficacy were evaluated in homologous modelsof human retinal disease. Because of the proliferative nature of hEScells, evidence of safety under Good Laboratory Practice (“GLP”)conditions is imperative for translating hESC-derived cellular productsinto the clinic. The extensive characterization detailed above providesassurance of cellular identity, whereas the long-term tumorigenicitystudy presented here provides strong evidence that the hESC-RPE cellsare safe and do not form teratomas and/or tumors during the lifetime ofNIH III immune-deficient mice. After introduction to the subretinalspace of RCS rats, the hESC-derived cells also survived for more than 8months without evidence of pathological consequences.

The hESC-RPE cells produced according to the methods described hereinalso rescued visual functions in a dose-dependent fashion: withincreased cell concentrations from 5,000 to 50,000, there was animprovement in functional rescue measured with both visual acuity andluminance threshold response. From 50,000 to 100,000, there is tolerancein numbers of cells introduced and that twice the optimal dose is stilleffective. Previous rodent work has shown that RPE cells quicklydisperse as a single or double layer and that 20,000 cells of animmortalized RPE cell line may occupy about 20% of the retinal area(12.56 mm²). For the age-related macular degeneration retina, the innermacular is 3 mm in diameter: this would mean that a dose of about 40,000cells may be used to cover the inner macular area but that a larger cellnumber may likely cover a larger area.

The significantly improved visual performance in Elovl4 mice adds to thevalue of the hESC-RPE as the cell choice for cell-based therapy to treatmacular disease (in this case, a subset of patients with Stargardt'sdisease caused by mutation in the Elovl4 gene). Stargardt's disease isone of the most frequent forms of juvenile macular degeneration.Although some rescue may be achieved by growth factor delivery such asdirect injection or factor-releasing cells (encapsulated cells) such asARPE19 cells transduced to produce ciliary neurotrophic factor orSchwann cells, these approaches cannot replace the other functions ofRPE cells. The hESC-RPE cells have a molecular profile more closelyresembling native RPE than do ARPE-19, and thus they may be able to takeon a broader range of RPE functions than ARPE-19 beyond simple factordelivery. For example, without being bound to a particular mechanism,the hESC-RPE cells may replace crucial functions of the host RPE becausethe hESC-RPE cells are able to phago-cytose latex beads in vitro.However, because the location of photoreceptor rescue extends beyond thearea of donor cell distribution, part of the rescue effect may bemediated by a diffusible trophic factor effect.

These results show the long-term safety of hESC-derived RPE cells inimmune-deficient animals, as well as their long-term function in twodifferent animal models of disease using GMP conditions suitable forclinical trials. The presence of differentiated human retinal pigmentedepithelial cells was identified incorporated or attached to the retinalpigmented epithelial cell layer of rats over 200 days post surgery. Inall cases, the morphology of these human cells was characterized asorganized cuboidal epithelial cells with round nuclei displaced by smallgolden-brown intracytoplasmic pigment, consistent with pigmentedepithelial cells. When associated with the mouse RPE, the human cellsdisplayed typical polarity along a basement membrane with basallylocated nuclei and apically located pigmented granules (FIGS. 13,14).The human cells could be distinguished from mouse RPE as the human cellsappeared slightly larger with fewer and smaller yellow-brown pigmentedgranules compared to the mouse RPE. Thus at P240 (i.e., 220 days aftertransplantation), donor cells survive, photoreceptors are rescued, and alevel of visual function is preserved. Thus, the methods describedherein may serve as a safe and inexhaustible source of RPE cells for theefficacious treatment of a range of retinal degenerative diseases.

Example 23 Rescue of Visual Function Using RPE Cells From Embryonic StemCells

Summary

Human embryonic stem cell-derived retinal pigmented epithelium (RPE)cells were assessed for their ability to retard the progression ofretinal degeneration in the Royal College of Surgeons (RCS) rats, a wellcharacterized and studied rodent model for retinal degeneration. Theseanimals carry a mutation in the gene for the MER tyrosine kinase(MERTK), which compromises the ability of RPE to perform phagocytosis ofshed photoreceptor outer segments. This dysfunction of RPE cells leadsto a progressive loss of both rods and cones overtime. Interestingly,mutation within the human orthologue of MERTK results in retinaldegeneration, whereby patients exhibit progressive poor visual acuityand visual field losses with age.

RPE cells were subretinally injected in RCS rat eyes at an early stageof retinal degeneration (P21) in order to prevent disease progression.Animals were divided into three groups: cell-injected group, balancedsalt solution (BSS)-injected control and untreated eyes. Cells (50,000,75,000 and 100,000 cells) were injected using BSS as the vehicle forcell delivery. For immune suppression cyclosporine was added to drinkingwater (210 mg/L) during the study. The efficacy of RPE cell injectionwas evaluated by two visual functional tests: optomotor responses andluminance threshold recordings from the superior colliculus (SC),followed by morphological examination including cresyl violet staining(for general retinal lamination and photoreceptor thickness).Additionally, immunostaining was performed with antibodies to humannuclei or human mitochondria antibodies to identify surviving humancells and the human RPE-specific marker bestrophin to their RPEphenotype. Both BSS injection alone and untreated eyes were used ascontrol groups these were examined along with cell injected groups atall the time points.

Results—Optomotor Responses

Animals were tested for spatial visual acuity using an optometry testingapparatus (CerebralMechanics, Lethbridge, Canada) comprised of fourcomputer monitors arranged in a square, which projected a virtualthree-dimensional space of a rotating cylinder lined with a verticalsine wave grating. Unrestrained animals were placed on a platform in thecenter of the square, where they tracked the grating with reflexive headmovements. The spatial frequency of the grating was clamped at theviewing position by recentering the cylinder on the animal's head. Theacuity threshold was quantified by increasing the spatial frequency ofthe grating using a psychophysics staircase progression until thefollowing response was lost, thus defining the acuity.

All cell-injected and control rats were tested from P60 to P240, the P60time point was chosen as the earliest time point when difference betweencell injection and control can be detected.

Cell-injected animals performed significantly better than BSS injectedand untreated controls at all time points tested (p<0.01). The majorityof the cell-injected animals had visual acuity above 0.5 cycle/degree atP90, which is similar to the visual acuity that non dystrophic rats(0.52-0.60 c/d)(8), while in BSS-injected and untreated control animals,an average of 0.25-0.30 c/d was recorded

Luminance Threshold Recording from the Superior Colliculus

This test is similar to the Humphrey test used in clinic for visualfield analysis in humans. In the case of animals, electrodes areimplanted and are measured using sensitive instrumentation. To assessluminance thresholds, single and multi-unit activity in the superficiallayers of the super colliculus (SC) was recorded.

Recordings were made from the superficial layers of the SC to a depth of100-300 μm using glass-coated tungsten electrodes (resistance: 0.5 MΩbandpass 500 Hz-5 KHz). Small craniotomies of about 100 μm in diameterwere made to access the brain. Anatomically, retinal ganglion cellsproject to contra-lateral superior colliculus (SC), therefore, right eyesends signals to left side of the SC. In the non dystrophic rat retina,there are 10-12 layers of photoreceptors which are very sensitive tolight stimuli. In other words, normal retina will respond to very lowlight stimulation, so normal animals have low luminance threshold(0.2-0.4 log units). In the dystrophic rat retina at P90, due to loss ofphotoreceptors, animals will only respond to high intensity lightstimulation, therefore these animals have a high luminance threshold(2.5-3.0 log units). Since the unit is expressed as logarithmic scale,0.2 log units in a normal retina is more than 100 times more sensitiveto light than 2.5 log units in a dystrophic retina.

Lower luminance thresholds were recorded in cell-injected eyes comparedwith BSS alone and untreated control eyes. Several of the cell-injectedeyes had luminance thresholds of 0.7-0.8 log units, compared with 3.0log units in untreated fellow eye (over 100 times more sensitive tolight stimulation).

The luminance threshold recorded from the SC correlated well with theamount of photoreceptors in the retina. Animals with more photoreceptorswere more sensitive to light stimulation, i.e. had a lower luminancethreshold. For example, one rat had extensive photoreceptorpreservation, which correlated with donor cell distribution. Optomotorresponse revealed visual acuity of 0.50 c/d compared with 0.25 c/d inuntreated eye, and luminance threshold recording gave a figure of 0.8log units at P90, compared with 3.0 log units in untreated control,which is more than 100 times more sensitive to light stimulation.

Histology

General Retinal Lamination

At the termination of the experiment, all animals were sacrificed bysodium pentobarbital overdose and perfused with phosphate-bufferedsaline. The eyes were removed and immersed in paraformaldehyde for onehour, infiltrated with sucrose, embedded in OCT medium and cut intohorizontal cryosections. All the retinal sections from cell-injected,BSS-injected and untreated controls were stained with cresyl violet forgeneral retinal lamination, identifying the injection site. There was noevidence of abnormal growth, teratoma formation or any other unwantedpathology.

HES-RPE Cell Survival

To confirm survival of human donor cells in rat eyes, the sections weredouble stained with anti-human mitochondria and anti-human bestrophinantibodies. Frozen eye sections were not originally intended for harshantigen retrieval procedures required for anti-human mitochondriastaining; thus a large number of sections was lost (came off the slidesfully or partially, resulting in poor morphology). The assay was furtheroptimized allowing double staining for anti-human nuclei and bestrophinwith good preservation of eye morphology. RPE cells were confirmed aspresent in 13 of 34 animals (38%). The majority of human cells (all butone animal where RPE cells were found in the intravitreous cavity) werefound at long term survival endpoints (P180-249), integrated into ratRPE layer, and all had typical RPE morphology and were positive for theRPE marker bestrophin which confirms the survival and preservation ofRPE identity at long-term post-transplantation in vivo.

Photoreceptor Preservation and Donor Cell Distribution

In cell-injected retina, there were 3-6 layers of photoreceptorscompared with localized 1-2 layers of photoreceptors around injectionsite in BSS control injection or a single layer of photoreceptors inuntreated retina at P90 thus pointing to photoreceptor preservationbeing associated with transplanted RPE cells. In BSS-injected eyes, 1-2cells thick localized rescue of photoreceptors was observed adjacent toinjection site around P90-100; however the effect was no longer evidentat later time points examined in this study. Luminance thresholdrecording also revealed this effect (usually one point had a lowerluminance threshold) two months after injection. With time, the effectof BSS injection disappeared while in cell-injected retina photoreceptorpreservation was seen out to P249 (over 225 days post-injection). inaddition, the secondary pathology related to progressive degenerationwas largely prevented, while in BSS injected and untreated retinas,typical secondary changes including vascular pathology and inner retinalneurons migrating into inner retina were clearly evident. Human specificantibody staining revealed hRPE cells surviving for over 225 dayspost-injection. The distribution of hES-RPE cells correlated withpreserved photoreceptor.

Conclusion

In all the retinas examined in this example, long-term preservation ofboth morphology and function after cell injection was demonstrated. TheRPE cells survived for at least 225 days, integrated into rat RPE layerand expressed the RPE cell specific marker bestrophin. No evidence ofunwanted overgrowth or teratoma formation was found. Therefore, the RPEcells described herein may be transplanted where they survive, maintaintheir phenotype, and rescue visual acuity in retinal degeneration.

Example 24 Treatment of Patient with Diabetic Retinopathy

A human patient diagnosed with diabetic retinopathy may be treated byadministering a pharmaceutical preparation comprising at least about100,000 human RPE cells (e.g., 100,000 RPE cells in 50 μL). The RPE cellpreparation is injected into sub-retinal space. The patient is placed ona treatment course of 5 mg/kg cyclosporin for 6 weeks. The patient ismonitored for the development of side effects. The visual acuity of thepatient is monitored and tested at least for 6 months followingtreatment.

Example 25 Treatment of Patient with Age-Related Macular Degeneration

A human patient diagnosed with age-related macular degeneration may betreated by administering a pharmaceutical preparation comprising atleast about 100,000 human RPE cells (e.g., 100,000 RPE cells in 50 μL).Prior to transplantation, the RPE cells may be cultured under conditionsthat increase alpha-integrin subunit expression. The RPE cellpreparation is injected into sub-retinal space. The patient is placed ona treatment course of 5 mg/kg cyclosporin for 6 weeks. The patient ismonitored for the development of side effects. The visual acuity of thepatient is monitored and tested at least for 6 months followingtreatment.

Example 26 Treatment of Patient with Retinal Pigmentosa

A human patient diagnosed with retinal pigmentosa may be treated byadministering a pharmaceutical preparation comprising at least about100,000 human RPE cells (e.g., 100,000 RPE cells in 50 μL). The RPE cellpreparation is injected into sub-retinal space. The patient is placed ona treatment course of 5 mg/kg cyclosporin for 6 weeks. The patient ismonitored for the development of side effects. The visual acuity of thepatient is monitored and tested at least for 6 months followingtreatment.

Example 27 Treatment of Patient with Stargardt's Disease

A human patient diagnosed with Stargardt's Disease (fundusflavimaculatus) may be treated by administering a pharmaceuticalpreparation comprising at least about 100,000 human RPE cells (e.g.,100,000 RPE cells in 50 μL). The RPE cell preparation is injected intosub-retinal space. The patient is placed on a treatment course of 5mg/kg cyclosporin for 6 weeks. The patient is monitored for thedevelopment of side effects. The visual acuity of the patient ismonitored and tested at least for 6 months following treatment.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. U.S. Provisional Patent Application Nos. 60/998,766,filed Oct. 12, 2007, 60/998,668, filed Oct. 12, 2007, 61/009,908, filedJan. 2, 2008, and 61/009,911, filed Jan. 2, 2008, the disclosures ofeach of the foregoing applications are hereby incorporated by referencein their entirety. In addition, the disclosure of WO 2009/05 1671 ishereby incorporated by reference in its entirety.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method of producing a pharmaceutical preparation or acryopreserved composition of human RPE cells comprising (a) culturinghuman pluripotent stem cells under non-adherent conditions for 7-14 daysto form cell masses; (b) culturing the cell masses to form an adherentculture comprising human RPE cells; (c) dissociating human RPE cellsfrom the adherent culture; (d) culturing said dissociated RPE cells toabout 95-100% confluency and a medium pigmentation; (e) dissociating thehuman RPE cells of (d); (f) repeating steps (d) to (e) at least once;(g) harvesting a population of said dissociated human RPE cells; (h)detecting expression of (i) ZO-1 and (ii) Pax6 and/or MITF in an aliquotof the harvested population to partially characterize the harvestedpopulation; and (i) formulating at least a portion of the harvestedpopulation as a pharmaceutical preparation or a cryopreservedcomposition of RPE cells if the harvested population is characterized as(1) ≥95% of the cells in the harvested population are positive for PAX6and/or MITF by immunostaining; and/or ≥95% of the cells in the harvestedpopulation are positive for PAX6 and/or bestrophin by immunostaining;and (2) ≥95% of the cells in the harvested population are positive forZO-1 by immunostaining; and (3) the harvested population is negative forOct-4, Nanog, Rex-1 and Sox2 by qPCR; and (4) the harvested populationis negative for Oct-4 and alkaline phosphatase by immunostaining;wherein RPE cells of the harvested population survive in vivo at leastabout 6 months, and wherein the culture conditions are not permissive topluripotent stem cells.
 2. The method of claim 1, wherein said humanpluripotent stem cells are selected from human induced pluripotent stem(iPS) cells, human embryonic stem (ES) cells, human adult stem cells,hematopoietic stem cells, fetal stem cells, mesenchymal stem cells,postpartum stem cells, multipotent stem cells or embryonic germ cells.3. The method of claim 1, wherein in step (e) the human RPE cells aredissociated using an enzyme selected from trypsin, collagenase, dispase,papain, a mixture of collagenase and dispase, and a mixture ofcollagenase and trypsin.
 4. A pharmaceutical preparation orcryopreserved composition comprising human RPE cells prepared by themethod of claim
 1. 5. The method of claim 1, wherein the human RPE cellsin the harvested population are (1) positive for bestrophin, RPE-65,CRALBP, PEDF, PAX6 and MITF by qPCR, and/or (2) positive for bestrophin,CRALBP, PAX6, MITF and ZO-1 by immunostaining.
 6. The method of claim 1,wherein greater than or equal to 85% of the human RPE cells in theharvested population are viable.
 7. The method of claim 1, wherein atleast a portion of the harvested population is formulated as acryopreserved composition and wherein greater than or equal to 70% ofthe human RPE cells therein are viable after cryopreservation.
 8. Thepharmaceutical preparation or cryopreserved composition of claim 4,wherein greater than or equal to 85% of the human RPE cells are viableprior to cryopreservation and/or greater than or equal to 70% of thehuman RPE cells are viable after cryopreservation.
 9. The pharmaceuticalpreparation or cryopreserved composition of claim 4, wherein the humanRPE cells are provided as a suspension of cells.
 10. The pharmaceuticalpreparation or cryopreserved composition of claim 4, wherein the humanRPE cells are provided in a pharmaceutical preparation and are disposedon a matrix.
 11. The method of claim 1, wherein the method steps areconducted in accordance with Good Manufacturing Practices (GMP) or GoodTissue Practices (GTP).
 12. The method of claim 1, wherein thedissociated RPE cells in (d) are cultured in RPE-GM.
 13. The method ofclaim 1, wherein the dissociated RPE cells in (d) are cultured inRPE-MM.